Applied and Environmental Microbiology最新文献

Environmental surveillance (ES) for poliovirus is a critical component of global eradication efforts, enabling the detection of virus circulation in communities, even in the absence of acute flaccid paralysis (AFP) cases. To monitor poliovirus circulation in a high-risk setting, ES was established in 2016 in Haïti in two cities (Port-au-Prince and Gonaïves) and expanded to five cities, adding Saint Marc, Cap Haïtien, and Port-de-Paix by 2023. Wastewater samples were collected monthly from 13 sites in urban areas and processed at CDC-Atlanta using the concentration and filtration elution (CaFÉ) method developed to concentrate enteroviruses, including polioviruses. Virus isolation was performed using poliovirus-sensitive (L20B) cells and enterovirus-sensitive (RD) cells, followed by molecular detection screening via real-time reverse transcription PCR designed for enterovirus and poliovirus typing. Between 2020 and 2023, sporadic Sabin-like poliovirus vaccine strains of serotypes 1 and 3 were detected, and no wild poliovirus (WPV) or vaccine-derived poliovirus (VDPV) was identified. The overall enterovirus detection rates ranged from 36% to 91% in 2020, 12% to 58% in 2021, 16% to 60% in 2022, and 27% to 73% in 2023, with some detection rates at some sites much lower than the optimal 50% target for global ES sites. Based on the lower detection rate, used as the main quality indicator for optimal ES, two sampling sites were discontinued in 2023 (one in Saint Marc and one in Cap Haïtien). The findings underscore the importance of high-performing ES in high-risk regions to augment AFP surveillance and serve as an early warning indicator of poliovirus circulation.IMPORTANCEPoliovirus environmental surveillance (ES) is a critical tool for detecting virus circulation before symptomatic cases of acute flaccid paralysis occur, especially in areas with inadequate surveillance. Haïti, a high-risk country for poliovirus transmission, faced numerous challenges from 2020 to 2023 that impacted ES operations, including political instability, humanitarian crisis, and site closures. This study provides a comprehensive evaluation of ES performance during that period, offering insights into surveillance resilience and sustainability in low-resource settings. The findings are timely and relevant, particularly in the context of the recent circulating vaccine-derived poliovirus type 2 (cVDPV2) outbreaks in Europe and Africa, and contribute to optimizing ES strategies globally to support polio eradication efforts.

Acetic acid bacteria such as Acetobacter spp. produce high concentrations of acetic acid by oxidizing ethanol while reducing molecular oxygen to water on the cell surface. The acetic acid resistance of acetic acid bacteria has been explained as a complex of several mechanisms. The aarC gene, encoding succinyl-CoA:acetate CoA transferase, which catalyzes the committed step of acetate metabolism, plays a crucial role in acetic acid resistance in Acetobacter aceti. Here, we constructed a mutant of Acetobacter pasteurianus strain SKU1108, from which aarC and its paralog aarC2 were deleted (named the ∆∆ strain). The ∆∆ strain failed to grow on a glucose- and glycerol-containing medium in the presence of acetic acid. However, when the ∆∆ strain was cultivated with ethanol, it grew well and produced acetic acid at comparable levels to the parental strain. When ethanol was added to growing cell-based acetic acid resistance experiments, the ∆∆ strain grew even in the presence of acetic acid, suggesting an ethanol-dependent but AarC-independent mechanism of acetic acid resistance. 2-Propanol and lactic acid were also oxidized by the ∆∆ strain and improved acetic acid resistance, though not as much as ethanol. In addition to growing cell assays, we established a resting cell-based cell killing assay with acetic acid. A protonophore, carbonyl cyanide m-chlorophenyl hydrazone, inhibited the ethanol-dependent acetic acid resistance of the ∆∆ strain in the cell killing assay. Thus, overall, we find that A. pasteurianus has AarC-independent but ethanol-dependent acetic acid resistance that is protonophore-sensitive.

Importance: Vinegar is produced by acetic acid fermentation by acetic acid bacteria such as Komagataeibacter spp. and Acetobacter spp. Resistance to acetic acid is an important feature of these microorganisms. At least two mechanisms have been proposed for acetic acid resistance: acetate metabolism and acetic acid efflux. The gene aarC is crucial in the acetate metabolism of Acetobacter sp. Here, a mutant derivative of Acetobacter pasteurianus strain SKU1108, devoid of aarC, failed to grow on acetate. In the absence of ethanol, the mutant was sensitive to acetic acid. However, in the presence of ethanol, it was resistant to acetic acid. These observations suggest a novel mechanism of AarC-independent but ethanol-dependent acetic acid resistance of A. pasteurianus. We can now find acetic acid-resistant mechanisms independent of acetate metabolism in growing cell-based experiments, which may promote elucidation of mechanism(s) of acetic acid resistance that involve as-yet-unidentified molecules.

Acrylonitrile-butadiene rubber (NBR), which is a petroleum-derived synthetic copolymer composed of acrylonitrile and 1,3-butadiene, is used in large amounts around the world for disposable gloves and oil seals. Most of which are incinerated without being reused. We aimed to develop a bio-upcycling method for waste NBR (wNBR) and studied microorganisms capable of assimilating NBR. An NBR-degrading actinomycete, Gordonia sp. strain J1A, which degraded 6.5%-11% in weight loss of a wNBR sample in 10 days, was isolated from an activated sludge of an industrial wastewater treatment plant. We have found a membrane-bound nitrile rubber oxygenase (Nro1) as a key enzyme that catalyzes the initial reaction step of NBR decomposition. Nro1 showed high homology to the amino acid sequences of MpaB family proteins, but no similarity to those of natural rubber-degrading enzymes (latex clearing proteins: Lcps). The degradation products, such as 4-cyano-1-cyclohexene and C22-C58 NBR oligomers, containing aldehyde or ester, as well as carbon black, were detected through the enzymatic degradation of the wNBR sample by Nro1. These findings offer a new strategy for a bio-upcycling method of wNBR.IMPORTANCEAn actinomycete, Gordonia sp., utilized multiple acrylonitrile-butadiene rubber (NBR)-degrading enzymes to cleave the main-chain carbon-carbon (C-C and C=C) bonds of NBR, a petroleum-derived synthetic rubber. The degradation products, such as 4-cyano-1-cyclohexene and C22-C58 NBR oligomers, containing aldehyde or ester, were detected through the enzymatic degradation of a waste NBR (wNBR) sample. One of the NBR-degrading enzymes, nitrile rubber oxygenase (Nro1), showed high homology to the amino acid sequences of MpaB family proteins. This study provides new insights into the enzymatic degradation of the petroleum-derived synthetic polymers.

Among environmentally widespread Sphingobacterium species, the animal feces isolate Sphingobacterium detergens E70 exhibited multidrug antibiotic resistance despite carrying few annotated antibiotic resistance genes (ARGs), prompting investigation of non-canonical mechanisms. This observation aligns with comparative genomic analysis of 62 Sphingobacterium genomes, which revealed only two to four ARGs per strain, typically associated with macrolide, phenicol, or tetracycline resistance. Remarkably, strain E70 lacked canonical ARGs yet displayed high-level resistance across nine antibiotics, including β-lactams and polymyxins. Scanning and transmission electron microscopy identified polymyxin B (PMB) as the most potent envelope-disrupting agent, revealing extensive outer-membrane vesiculation following PMB exposure. Fluorescence-based cytometric and microscopic analyses revealed PMB-induced phenotypic alterations, characterized by enhanced biofilm formation, modified surface charge, and changes in lipid composition. Inhibition of sphingolipid biosynthesis with myriocin markedly reduced vesiculation and impaired growth, indicating that sphingolipids contribute to envelope integrity and stress adaptation under PMB treatment. Confocal imaging with dansyl-labeled PMB showed that myriocin-mediated sphingolipid depletion made the membrane more permeable to PMB and reduced surface fluorescence, reflecting altered membrane environments. Together, these findings uncover a previously unrecognized role of sphingolipids in PMB resistance, that is, by promoting outer membrane vesicle-mediated membrane remodeling, sphingolipids enhance PMB resilience in S. detergens E70 and potentially in other sphingolipid-producing bacteria.

Importance: Environmental bacteria often display antibiotic tolerance without carrying canonical resistance genes. We show that Sphingobacterium exploits sphingolipid-dependent outer membrane vesiculation as a structural defense against polymyxin B. Blocking sphingolipid biosynthesis with myriocin suppressed vesiculation and sensitized cells to polymyxin B, indicating that these rare bacterial lipids provide essential sites for drug interaction and membrane remodeling. Our findings reveal a lipid-driven mechanism of vesiculation and highlight sphingolipid metabolism as a potential therapeutic target.

In situ remediation of groundwater at coal combustion product (CCP) sites can be challenging for elements such as molybdenum (Mo), which do not respond well to commonly used treatment. This research was initiated to improve the understanding of geochemistry and microbial diversity associated with a Mo plume at a CCP site toward the development of an in situ treatment scheme. Diffusive microbial samplers were designed and deployed at the study site for 9 weeks. Afterward, geochemical and community analyses were used as the basis to understand how microbial communities respond to elevated Mo concentrations within a plume. Our results show that the Mo and other constituents within the plume do not reduce the diversity of the community, in contrast to trends observed at other industrial sites with metals and metalloids in groundwater. Interestingly, bacteria of the order Burkholderiales were higher in abundance in wells where Mo >0.3 mg/L, and several sulfate-reducing bacteria were less abundant but not absent. Molybdenum sequestration experiments were also performed with sulfate-reducing bacteria enriched from groundwater samples collected at the site. The results show that Desulfomicrobium escambiense played a major role in Mo sequestration and activated a detoxification mechanism. This process involved the sequential activation of periplasmic heavy metal sensors, followed by the activation of atpE ATP synthase, which may function as an exporter of Mo to form Mo-S species in the periplasm of the cell. The results provide important considerations for bioremediation potential in groundwater settings impacted by Mo, especially those who seek to stimulate sulfate-reducing bacteria for Mo sequestration in biogenic sulfide solids.IMPORTANCEBioremediation of contaminated sites has become popular for chlorinated hydrocarbons, but it has not been widely applied to inorganic constituents outside of arsenic. Here, we show the potential for the development of geochemistry-informed bioremediation technologies of Mo-contaminated groundwater by leveraging Mo-tolerant communities despite the suppression of sulfate reduction by Mo.

Vibrio parahaemolyticus (V. parahaemolyticus) is a widespread marine bacterium that has gained prominence as a major seafood-borne pathogen and a primary cause of human gastroenteritis worldwide. The type VI secretion system (T6SS), a highly specialized molecular apparatus that injects effector proteins into competing cells, plays a pivotal role in mediating the pathogenicity, bacterial competitiveness, and environmental adaptability. This review presents an in-depth overview of the T6SS in V. parahaemolyticus, addressing its discovery, molecular architecture, genetic organization, and associated effector proteins. In addition, special attention is given to the functional divergence between T6SS1 and T6SS2, particularly in mediating bacterial antagonism and adaptation to environmental stressors. We further examine the complex regulatory frameworks that control these systems, including environmental signals, surface sensing, quorum sensing, and specific regulators. Understanding these mechanisms advances our knowledge of the survival strategies and pathogenic behaviors of V. parahaemolyticus.

Magnetotactic bacteria (MTB) biomineralize intracellular, membrane-enclosed magnetite or greigite nanocrystals (magnetosomes). How magnetosome gene clusters (MGCs) control magnetosome morphology and evolve across lineages remains central to reconstructing the history of magnetotaxis. Here, we report five uncultured MTB strains from Yuyuantan Lake (Beijing, China), all within Rhodospirillales order (Alphaproteobacteria class). Using phylogenetics, fluorescence in situ hybridization-scanning electron microscopy, and transmission electron microscopy, we show that magnetosome morphology is more strongly constrained by phylogeny than by cell morphology. Whole-genome comparisons and MGC phylogenies indicate that vertical inheritance predominates at the genus level, whereas topological incongruences reveal additional processes, notably horizontal transfer and gene duplication. In particular, the presence of a canonical mamAB operon together with a duplicated mamAB-2 cluster supports inter-genus horizontal gene transfer between Magnetospirillum and Paramagnetospirillum. These findings refine evolutionary models by showing that conserved MGC architectures provide a stable scaffold for magnetosome biomineralization while permitting diversification within the Alphaproteobacteria class.IMPORTANCEMagnetotactic bacteria (MTB) build intracellular magnetic nanoparticles (magnetosomes) that guide navigation and influence biogeochemical cycling. Yet how the underlying genes map onto ancestry and crystal shape remains unclear. Pairing quantitative crystal-morphology statistics with phylogenomic analysis for MTB from the Rhodospirillales order, we show that magnetosome traits carry a stronger phylogenetic signal than cell shape. Newly recovered uncultured strains broaden Paramagnetospirillum diversity, and a high-quality genome (YYTV-2) represents a novel species within the rarely studied Candidatus Magneticavibrio. Analyses of both the canonical mamAB operon and a duplicated mamAB-2 cluster indicate predominantly vertical inheritance, with horizontal transfer and gene duplication introducing modular variation. These results tighten genotype-mineral phenotype links, improving the interpretation of magnetofossils and MTB as indicators of environmental change.

Salmonella enterica is a leading bacterial cause of foodborne illness, often transmitted through contaminated food and water. Improved food handling has led to considerable reductions of Salmonella contamination in meat and poultry products; this does not wholly contribute to decreased salmonellosis incidence, highlighting the need to define alternative reservoirs and transmission pathways. In this study, we collected samples from four distinct watersheds over 24 months to characterize Salmonella serovar diversity and utilized phylogenetic approaches, along with proximal land use analyses, to identify relationships between environmental reservoirs and hosts. Across 19 sites, including animal agriculture, suburban, and forested areas, 10 L water samples were collected (n = 456), and cultured for Salmonella, followed by whole genome sequencing of isolates and deep serotyping of positive samples. Overall prevalence was 69% (314/456), and generalized linear mixed models showed that compared to proximal land use, seasonal weather patterns, including precipitation and humidity, significantly influenced recovery and complexity. Antimicrobial resistance was detected in 11% (33/314) of isolates, with 21% (7/33) classified as multidrug resistant. CRISPR-SeroSeq identified 37 serovars, and multiserovar populations were detected in 89% (229/258) of positive samples with sequencing data, averaging 3.7 serovars/sample. Comparison with national food animal production monitoring showed limited serovar overlap, with serovar Rubislaw dominating water samples but absent in agricultural data sets. Collectively, these results demonstrate extensive serovar diversity within Salmonella populations in freshwater systems, including clinically relevant serovars, and emphasize the need to develop a robust surveillance platform for source attribution and, ultimately, prevention of future outbreaks.IMPORTANCEContaminated surface water significantly contributes to global Salmonella illnesses, marking a critical need to assess serovars present and determine environmental variables affecting the population dynamics in this reservoir. We found that complex multiserovar populations, often including pathogenic serovars, occur in surface water regardless of proximal land use. Notably, many aquatic serovars are not detected in animal agriculture monitoring and the phylogeny presented here shows isolates are more closely related to human clinical than animal-source isolates. However, limited serotyping data are available for alternative reservoirs of foodborne illness, namely, wildlife, which hinders source attribution. This study highlights a significant gap in understanding environmental Salmonella transmission and underscores the importance of a One Health surveillance approach to protect public health.

Bacterial metabolites are essential for biological processes, influencing human health, ecosystems, and various industrial applications. Simultaneous real-time monitoring of these metabolites is critical in understanding microbial dynamics, particularly in bioreactors and food or drug manufacturing. Current approaches often rely on offline methods, which are labor-intensive and susceptible to contamination, or genetic engineering techniques limited to single-analyte monitoring. Here, we present a novel method utilizing engineered periplasmic binding proteins (PBPs) conjugated with fluorophores to track multiple metabolites simultaneously in bacterial cultures, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Clostridium striatum. This system continuously monitors the levels of multiple analytes, such as glucose, arabinose, ribose, glutamate, and arginine, providing high temporal resolution while maintaining sensor stability over a period of 24 h. Our findings confirm hierarchical substrate utilization in E. coli and other bacterial species and demonstrate the versatility of PBP-based multi-sensor arrays. This approach offers a non-invasive, modular, and scalable tool for bacterial metabolite analysis, paving the way for advances in both fundamental discoveries and practical applications in microbiology.

Importance: Real-time monitoring of metabolites in bacterial cultures is crucial for advancing our understanding of microbial physiology, metabolic fluxes, and dynamic responses to environmental changes. This capability enables researchers to capture transient metabolic states that are often missed in endpoint measurements. The use of engineered periplasmic binding proteins as biosensors for this real-time metabolite monitoring represents a groundbreaking approach. By leveraging the natural specificity and high affinity of PBPs for small molecules, these biosensors can be engineered to detect a wide range of metabolites with exceptional sensitivity and temporal resolution. The integration of PBP-based biosensors into microbial research not only enhances our ability to study real-time metabolism but also provides a versatile tool for optimizing industrial bioprocesses and exploring bacterial infections and complex microbial ecosystems.