Applied and Environmental Microbiology最新文献

{"title":"Engineering a T7 bacteriophage to attenuate LPS-driven inflammatory responses during bacteriolysis.","authors":"Tong Yu, Junjiao Pang, Mengge Chen, Qi Sun, Jiaqi Pu, Deshu Wang, Qingling Liu, Fengtang Yang, Hongkuan Deng","doi":"10.1128/aem.02303-25","DOIUrl":"https://doi.org/10.1128/aem.02303-25","url":null,"abstract":"<p><p>Bacterial lysis during treatment of Gram-negative infections can release lipopolysaccharide (LPS) and aggravate inflammation. Here, we engineered two complementary T7 bacteriophages: T7-<i>nluc</i>, a NanoLuc reporter bacteriophage for real-time monitoring of viable bacteria, and T7-<i>phoa</i>, a therapeutic bacteriophage that releases alkaline phosphatase (PhoA) during lysis to reduce LPS bioactivity. Both engineered bacteriophages retained lytic activity similar to that of wild-type T7. <i>In vitro</i>, T7-<i>nluc</i> produced a low-background bioluminescent signal that reflected bacterial burden, whereas T7-<i>phoa</i> released catalytically active PhoA into the extracellular environment. In <i>Galleria mellonella</i> and <i>Danio rerio</i> infection models, T7-<i>nluc</i> enabled dynamic monitoring of infection progression, while T7-<i>phoa</i> improved survival, reduced inflammatory responses, and accelerated inflammatory resolution without compromising bacterial clearance. These findings support a modular bacteriophage engineering strategy that combines bacterial killing, real-time infection monitoring, and local attenuation of LPS-driven inflammation, offering a potential approach for improving bacteriophage-based treatment of Gram-negative infections.</p><p><strong>Importance: </strong>Bacteriophage therapy is being reconsidered for treating drug-resistant Gram-negative infections, but there is concern that rapid bacterial lysis may release LPS and worsen inflammation. We used bacteriophage T7 as a platform to test whether bacteriophages can be engineered to both fight bacteria and soften these harmful host responses. First, we created a NanoLuc reporter bacteriophage that produces light only when it grows in live bacteria, confirming that engineered bacteriophages can deliver active proteins directly in infected animals. We then built a therapeutic T7-<i>phoa</i> bacteriophage designed to release enzymatically active alkaline phosphatase upon on-target lysis, thereby providing lysis-coupled local phosphatase activity at the infection site. In both <i>G. mellonella</i> and <i>Danio rerio</i> models, infection-site fluids collected after treatment showed elevated phosphatase activity in the T7-<i>phoa</i> group, and the treatment was associated with lower inflammatory peaks, improved survival, and preserved bacterial clearance. Together, these results support a modular route for bacteriophage-based strategies that couple bacterial killing with real-time reporting and local control of LPS associated inflammation.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0230325"},"PeriodicalIF":3.7,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147809987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recent advances in exploring the composition and evolution of the prokaryotic selenoproteome.","authors":"Yan Zhang, Shuting Wang, Hengtao Li, Xuan Chen","doi":"10.1128/aem.00556-26","DOIUrl":"https://doi.org/10.1128/aem.00556-26","url":null,"abstract":"<p><p>Selenoproteins, a unique class of proteins critical for cellular antioxidant defense, are characterized by the incorporation of selenocysteine (Sec) in their active sites. Sec is co-translationally inserted into proteins via a specialized mechanism that reprograms the UGA codon to encode Sec, involving a specific RNA structure designated the Sec insertion sequence (SECIS) element and several essential enzymes. Although numerous selenoproteins have been identified in prokaryotes (primarily bacteria), the detection of selenoprotein genes in these organisms remains challenging, largely due to difficulties in distinguishing the Sec-encoding UGA codon from standard termination signals. In recent years, computational approaches for predicting selenoprotein genes, along with comparative genomic analyses of Sec-encoding machinery and selenoproteomes, have emerged as a promising and rapidly evolving field, offering new insights into Sec utilization in bacteria and archaea. This review provides a comprehensive overview of the latest advancements in the study of selenoproteins in prokaryotes. We summarize the molecular mechanisms underlying Sec biosynthesis and incorporation, and the structural diversity of SECIS elements in bacteria and archaea. We then describe current computational strategies for the identification of prokaryotic selenoprotein genes and present an updated, extensive catalog of prokaryotic selenoproteins documented to date, emphasizing those with well-established functions. Finally, we discuss recent progress in understanding the evolutionary dynamics of the Sec-encoding system and selenoproteins across prokaryotes, with a focus on the archaea-to-eukaryote transition of Sec machinery and selenoproteins. Overall, this review offers a unified perspective on the identification, functions, and evolution of selenoproteins in prokaryotes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0055626"},"PeriodicalIF":3.7,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147809919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Persistence of protozoan parasites and <i>Escherichia coli</i> on plant tissue in soil.","authors":"Kyle J McCaughan, Michelle D Danyluk, Kalmia E Kniel","doi":"10.1128/aem.02467-25","DOIUrl":"https://doi.org/10.1128/aem.02467-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Tilling under is a method growers employ in an effort to remediate instances of widespread contamination in fields of crops, but its effectiveness with respect to specific pathogens remains poorly understood. This study had two overarching goals: first, to assess the survival dynamics of &lt;i&gt;Cryptosporidium parvum&lt;/i&gt; and &lt;i&gt;Eimeria tenella&lt;/i&gt; (used as a surrogate for &lt;i&gt;Cyclospora cayetanensis&lt;/i&gt;) oocysts, and &lt;i&gt;Escherichia coli&lt;/i&gt; TVS355 in soil following inoculation onto plant tissue and a simulated \"tilling under\" process; and second, to evaluate the potential of monitoring mRNA levels over time as oocysts die off as an alternative to traditional infectivity-based methods to determine oocyst viability. Basil, cilantro, and soil without plant tissue were inoculated with &lt;i&gt;C. parvum&lt;/i&gt; (10&lt;sup&gt;6&lt;/sup&gt; oocysts), &lt;i&gt;E. tenella&lt;/i&gt; (10&lt;sup&gt;6&lt;/sup&gt; oocysts), and &lt;i&gt;E. coli&lt;/i&gt; TVS355 (10&lt;sup&gt;8&lt;/sup&gt; CFU) and then incorporated into 120 g of soil; samples were then incubated at 12°C or 32°C for up to 12 months. Monthly, samples were removed and assessed for organism survival, &lt;i&gt;E. coli&lt;/i&gt; TVS355 through traditional dilution and plating, and oocyst decay through two methods: cell culture followed by qPCR, and mRNA extraction followed by RT-qPCR to measure gene expression. &lt;i&gt;E. coli&lt;/i&gt; remained detectable in samples for 10 months, and, through infectivity methods, &lt;i&gt;C. parvum&lt;/i&gt; and &lt;i&gt;E. tenella&lt;/i&gt; remained infectious for 9 months. However, results from mRNA methods indicated that oocysts remained detectable for at least 12 months, with approximately 1.22 log oocysts/sample detected at that point for both &lt;i&gt;C. parvum&lt;/i&gt; and &lt;i&gt;E. tenella&lt;/i&gt;. Temperature impacted &lt;i&gt;E. coli&lt;/i&gt; TVS355 and &lt;i&gt;E. tenella&lt;/i&gt; recovery (as determined by infectivity methods) only after 7 months, when slightly more of each organism was recovered from samples incubated at 12°C. Meanwhile, mRNA methods detected significantly (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) fewer oocysts of both &lt;i&gt;C. parvum&lt;/i&gt; and &lt;i&gt;E. tenella&lt;/i&gt; in samples incubated at 32°C as compared to those incubated at 12°C throughout the experiment.IMPORTANCEContaminated crops may be tilled under and buried in the soil, which may lead to the decay of contaminating microbes. This study looked at three microbes that can contaminate produce: &lt;i&gt;Escherichia coli&lt;/i&gt; (a bacterium), &lt;i&gt;Cryptosporidium parvum&lt;/i&gt; (a zoonotic protozoan pathogenic parasite), and &lt;i&gt;Eimeria tenella&lt;/i&gt; (an avian protozoan parasite used as a stand-in for the human parasite &lt;i&gt;Cyclospora cayetanensis&lt;/i&gt;). Inoculated basil and cilantro were mixed in soil and stored at 12°C and 32°C for 1 year. Both protozoan parasites remained infectious for 9 months, and &lt;i&gt;E. coli&lt;/i&gt; remained viable for 10 months. Protozoa genetic material and mRNA, as a sign of metabolic activity, were also assessed over time. The type of plant tilled under did not make a meaningful difference. Tilling contaminated crops into soil does not quickly eliminate pathogens.","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0246725"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Viral architects of the deep biosphere: reshaping the framework of sedimentary biogeochemistry.","authors":"Chong Wang, Chaomin Sun","doi":"10.1128/aem.00486-26","DOIUrl":"https://doi.org/10.1128/aem.00486-26","url":null,"abstract":"<p><p>A recent minireview by J. R. A. Williams and J. F. Biddle (Appl Environ Microbiol, 92:e00275-25, 2026, https://doi.org/10.1128/aem.00275-25) substantially reframes our understanding of sedimentary viruses. For decades, viruses in marine sediments have been viewed primarily as agents of mortality, their roles largely confined to the canonical \"viral shunt\" paradigm developed for pelagic systems. The authors expand this perspective, positioning viruses as active participants in benthic biogeochemistry-contributing to nutrient cycling, modulating microbial diversity, and influencing organic matter processing and carbon sequestration. This conceptual shift highlights sedimentary viruses as an integral and, until now, underappreciated component of global element cycles.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0048626"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interpretable convolutional neural networks for sequence-based classification and discovery of plastic-degrading enzymes.","authors":"Woo-Haeng Lee, Louis Dumontet, KyungMin Jung, Hyun Lee, Gobinda Thapa, Tae-Jin Oh, Mingon Kang","doi":"10.1128/aem.01586-25","DOIUrl":"https://doi.org/10.1128/aem.01586-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;The rapid accumulation of plastic waste has emerged as a critical environmental threat, driving the need for scalable and effective biodegradation solutions. Major plastic types, including biopolyesters, aliphatic polyesters, and aromatic polyesters, are widely used in industrial and consumer products and exhibit distinct chemical structures and degradability profiles, posing challenges for systematic enzyme classification. Hydrolytic plastic-degrading enzymes (PDEs) offer a promising solution, yet their functional classification remains limited by insufficient annotations and enzymatic diversity. In this study, we present an explainable deep learning framework, plastic-degrading enzyme prediction via interpretable CNN (PEPIC), to classify enzymes across nine plastic substrate types directly from protein sequences. Using a curated data set of experimentally validated plastic-degrading enzymes (181 sequences) and an expanded homologous data set generated via sequence similarity search (~5,900 sequences), we benchmarked PEPIC against state-of-the-art approaches and evaluated both predictive performance and interpretability. PEPIC demonstrated statistically significant improvements in F1-score compared to state-of-the-art methods. PEPIC calculated contribution scores at the amino acid level, indicating how individual residues influence the predictions. The model interpretation revealed that regions with high contribution scores aligned with key catalytic residues. Homology-based structural modeling demonstrated that residues with high contribution scores mapped to known catalytic and substrate-binding regions of plastic-degrading enzymes and reflected structural differences across plastic classes, supporting the biological relevance of PEPIC's predictions. PEPIC identified an uncurated enzyme as a potential PET-degrading candidate. This work provides an interpretable framework for plastic-degrading enzyme discovery, which can accelerate biotechnological solutions for plastic waste management and support data-driven strategies toward sustainable environmental remediation.IMPORTANCEWe propose an explainable deep learning-based approach, named PEPIC, that can effectively classify enzymes across nine plastic substrate categories relevant to hydrolytic PDE activity and provide trustworthy predictions by identifying active and binding sites that align with prior biological knowledge. PEPIC is the first study that demonstrated the high potential of deep learning-based approaches for plastic-degrading enzymes prediction using large data sets. PEPIC not only significantly improved predictive performance compared to the current state-of-the-art models but also provided the trustworthiness of the prediction. PEPIC was thoroughly assessed by intensive and comprehensive experimental settings, and PEPIC enhances the model interpretation for trustworthy predictions and potential new biological knowledge discovery. This work offers scientific advances in acce","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0158625"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Delftia</i> as a small-molecule chassis: lessons from delftibactin and harmane.","authors":"Pushkar Sai, Andrew Hoyek, Carlos C Goller","doi":"10.1128/aem.00277-25","DOIUrl":"https://doi.org/10.1128/aem.00277-25","url":null,"abstract":"<p><p><i>Delftia</i> spp. occur in diverse environmental and host-associated settings, but their small-molecule capabilities are not uniform across the genus. In this Minireview, we use a strain-resolved \"small-molecule chassis\" framework to examine cases in which exported, low-molecular-weight metabolites can be linked to extracellular phenotypes. We focus on two anchor systems: delftibactin, a siderophore-like nonribosomal peptide metallophore associated with Au(III) detoxification and gold biomineralization in <i>Delftia acidovorans</i>, and harmane, a β-carboline linked to inhibition of early <i>Plasmodium</i> development by <i>Delftia tsuruhatensis</i> TC1 in mosquitoes. These systems do not represent the same level of mechanistic resolution, with delftibactin providing the tighter genotype→metabolite→phenotype chain and TC1 spanning both a harmane-linked mosquito phenotype and a less resolved low-molecular-weight supernatant phenotype in sand flies. Comparative genomics further supports the idea that these activities are strain- and lineage-specific rather than genus-wide. We argue that the small-molecule chassis framework is most useful not as a blanket label for <i>Delftia</i>, but as a way to identify which strains justify deeper mechanistic study and cautious translation.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0027725"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Memoriam: Robert M. Kelly (1953-2026).","authors":"Michael W Adams, Jason M Haugh, Albert J Keung, Balaji M Rao, Sindee L Simon","doi":"10.1128/aem.00599-26","DOIUrl":"https://doi.org/10.1128/aem.00599-26","url":null,"abstract":"<p><p>The chemical and biochemical engineering, microbiology, and biotechnology communities deeply mourn the passing of Dr. Robert M. Kelly, the Alcoa Professor of Chemical and Biomolecular Engineering and Director of the Biotechnology Program at NC State University. A towering pioneer in the biology of extremophiles, an esteemed educator and mentor, and a dedicated editor for <i>Applied and Environmental Microbiology</i> (AEM), Bob leaves behind an extraordinary legacy that reshaped our understanding of life at high temperatures.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0059926"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Vibrio cholerae</i> O1 classical and El Tor toxin-coregulated pilus subunit A (TcpA) epitopes that induce biotype-specific and cross-protective antibodies against bacterial adherence.","authors":"Sai Simha Reddy Vakamalla, David A Sack, Weiping Zhang","doi":"10.1128/aem.00350-26","DOIUrl":"https://doi.org/10.1128/aem.00350-26","url":null,"abstract":"<p><p>The persistent and deadly cholera remains a significant burden on global health. Current countermeasures are inadequate against cholera. Toxin-coregulated pilin (TCP) has long been a target for understanding the biogenesis of <i>Vibrio cholerae</i> infection and for developing subunit vaccines against cholera. In this study, we examined TcpA B-cell epitopes of the O1 classical and El Tor biotypes, as well as the O139 serogroup, for functionality, potential biogenesis, and vaccine application. By empirically mapping TcpA functional epitopes across <i>V. cholerae</i> biotypes and serogroups, we found that antibodies raised against a shared epitope (EP #1, IQSQNMTKAAQ) provided partial protection against <i>V. cholerae</i> bacterial adherence <i>in vitro</i>. Data also showed that classical biotype TcpA epitope #6, ASKNLDLTNITHVE, is more effective in inducing functional antibodies against adherence of the classical biotype, but not against the El Tor biotype. Similarly, El Tor biotype TcpA epitopes #4 (FAAVADLGDFETSV) and #6 (GSANLNLTNITHVE) are effective in inducing functional antibodies against the adherence of El Tor biotype and O139 serogroup, but not against the classical biotype. This decodes early observations of a lack of cross-protection from TcpA-derived immunity. Interestingly, combined immunity raised against the classical epitope #6 and the El Tor epitope #6 (or #4) effectively protected against adherence from both biotypes. These results reveal the mechanism underlying the lack of protective immunity conferred by biotype-specific TcpA and indicate a combined epitope immunity for protection across biotypes and serogroups, providing a better understanding of TcpA immunity, perhaps its biogenesis, and guiding the development of cholera vaccines.IMPORTANCECurrent countermeasures are inadequate against cholera, a disease that remains a significant global health burden. Oral cholera vaccines (OCVs) prequalified by the World Health Organization (WHO) are serogroup-specific. In addition to the supply shortage, these whole-cell vaccines provide limited protection to children under 5 years of age in endemic countries, the most vulnerable population to cholera, urgently calling for more effective prevention strategies. By examining functional immunity raised against shared and biotype-specific TcpA epitopes, this study revealed the mechanism underlying why TCP-related immunity from the O1 classical biotype does not extend protection to the O1 El Tor biotype. The current study also showed that a combined biotype-epitope immunity provides cross-protection against O1 El Tor and classical biotypes, as well as against the O139 serogroup, potentially paving the way for the development of a cross-protective vaccine against <i>Vibrio cholerae</i> infection.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0035026"},"PeriodicalIF":3.7,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Water-soluble swab material for environmental sampling.","authors":"Kevin K Crown, Timothy N Lambert, Maria Kelly, Nelson S Bell, Joshua Santarpia","doi":"10.1128/aem.00030-26","DOIUrl":"https://doi.org/10.1128/aem.00030-26","url":null,"abstract":"<p><p>Biological threats represent a growing vulnerability to national security, affecting both military and civilian populations. Deliberate dissemination of chemical and biological (CB) agents can have immediate and significant impacts on public health and the economy, highlighting the need for faster and more efficient techniques to assess contamination and safely clear affected areas for public reentry following an accidental or intentional biological release. Current surface sampling procedures set forth by the Centers for Disease Control and Prevention (CDC) call for sampling swab materials that are not soluble in aqueous bacterial growth medium. These have been shown here to be inefficient, releasing less than 40% of the captured biological material into aqueous solution for detection. Since the CDC put these procedures in place, there has been no impetus for industry to develop sampling swabs that release 100% of the sample they capture from a non-porous surface. This project developed cost-effective, novel, biologically hospitable, adsorbent materials that dissolve in water, releasing 100% of the captured sample. These fibers maintain structural integrity during sampling, can be premoistened without premature dissolution, and rapidly dissolve in solution, facilitating efficient sample release. Forcespun PVA fibrous swabs demonstrated significantly higher spore capture and release efficiency compared to traditional materials, without inhibiting bacterial growth or spore germination. These swabs could significantly improve current sampling technologies and procedures via increased spore detection and analysis efficiency. Higher sampling efficiency could translate to fewer samples required for analysis and significantly less time to render an area safe for public reentry. Increased sampling efficiency would also provide faster impact data to decision makers and lead to improved consequence management. Prior to this study, water-soluble swabs suitable for this purpose have not yet been developed.IMPORTANCEAccurate environmental sampling is critical for detecting low-level microbial contamination in both public health and biodefense settings. Our water-soluble PVA swab material achieves up to 100% spore release from non-porous surfaces, more than double the recovery of traditional swabs, while being compatible with scalable, low-cost fabrication methods. This new tool will enable more sensitive, faster, and more reliable surface monitoring across a wide range of environmental and diagnostic applications.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0003026"},"PeriodicalIF":3.7,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Developing an optimized method for biofilm extraction from microplastic surfaces for high-efficiency analysis of adherent bacterial communities.","authors":"Hieu Hoai Vo, Thao Thanh Le, Tu Van Nguyen, Jennifer Scott, Tony Gutierrez, Michel J Kaiser, Huong Thi Thuy Ngo","doi":"10.1128/aem.00416-26","DOIUrl":"https://doi.org/10.1128/aem.00416-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Microplastics (MiPs, ×5 mm in size) harbor complex biofilms that facilitate pathogen dissemination, yet standardized extraction protocols are lacking. Here, we developed and optimized a method for biofilm extraction from environmentally weathered MiPs. To reflect real-world conditions, the protocol was applied directly to bulk, heterogeneous, field-collected MiP mixtures (size range: 80 µm-5 mm) without prior sorting by polymer type or morphology. By optimizing extraction buffers, mechanical disruption, and MiP quantities (100-150 particles), we established an optimal protocol combining phosphate-buffered saline with 0.1% Tween 80, ultrasonication (40 kHz, 10 min), vortexing with glass beads, and a two-cycle extraction-disaggregation workflow. This approach involves an initial extraction followed by a repeated, exhaustive extraction step designed to maximize the recovery of recalcitrant biofilm residues. This protocol markedly enhanced recovery of viable, culturable cells, delivering a 2,950-fold enhancement in the recovery of viable, culturable cells (evaluated via CFU counts; 28,020 ± 11,034 CFU MiP⁻¹) vs. conventional PBS extraction (9.5 ± 3 CFU MiP⁻¹) and 102-fold vs. passive extraction (274 ± 59 CFU MiP⁻¹). The 10-min sonication empirically maximized viable cell recovery within the tested duration range. The two-step protocol with Tween 80-mediated disaggregation proved critical, increasing recovery 208-fold by disaggregating biofilm fragments. While DNA yields (26.5 ± 3.93 ng µL⁻¹) were sufficient for targeted PCR-based pathogen detection (&lt;i&gt;Aeromonas&lt;/i&gt; spp., &lt;i&gt;Salmonella enterica&lt;/i&gt;), the co-extraction of complex environmental matrices (&lt;i&gt;A&lt;/i&gt;&lt;sub&gt;260&lt;/sub&gt;/&lt;i&gt;A&lt;/i&gt;&lt;sub&gt;280&lt;/sub&gt; ratio: 0.17-0.19) strictly requires an additional purification step prior to next-generation sequencing. Validation across contrasting aquatic environments confirmed the method's robustness. Comparative analysis demonstrates that conventional single-step approaches fail to recover the majority of viable cells trapped within weathered MiP biofilms. This optimized and validated protocol provides a critical methodological foundation for investigating plastisphere microbial ecology and pathogen transport dynamics, supporting evidence-based risk assessment of MiP contamination, especially public health risks associated with microplastic pollution.IMPORTANCEMicroplastic-associated biofilms (the \"plastisphere\") serve as vectors for waterborne pathogens and antibiotic resistance genes; however, the persistent use of inadequate extraction methods has systematically underestimated microbial abundance, presenting a critical barrier to global environmental risk assessment. By overcoming the limitations of conventional extractions-which fail to penetrate recalcitrant extracellular polymeric matrices on environmentally weathered microplastics-our standardized methodology liberates previously undetectable bacterial populations. The ability to accurately quantify the","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0041626"},"PeriodicalIF":3.7,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}