Applied and Environmental Microbiology最新文献

{"title":"Evaluation of <i>Staphylococcus simulans</i> inhibition of <i>Staphylococcus aureus</i> infection by an <i>in vivo</i> murine mastitis model.","authors":"Benjamin Caddey, Mengyue Li, Jeroen De Buck, Bo Han, Jian Gao, Herman W Barkema","doi":"10.1128/aem.00678-25","DOIUrl":"10.1128/aem.00678-25","url":null,"abstract":"<p><p><i>Staphylococcus aureus</i> is a major bovine mastitis pathogen and can result in chronic intramammary infections that are subject to considerable antimicrobial use. In comparison, non-<i>aureus</i> staphylococci (NAS) are common in intramammary infections, but some strains of NAS reduce risk of clinical mastitis and can inhibit <i>S. aureus</i> growth <i>in vitro</i>. This study aims to determine whether <i>in vitro</i> inhibition of <i>S. aureus</i> growth by NAS species translates to <i>in vivo</i> inhibition within a mouse mastitis model and to characterize NAS mammary tissue colonization. Two <i>Staphylococcus simulans</i> strains were selected for <i>in vivo</i> experimentation to compare genetically similar strains that can and cannot inhibit <i>S. aureus in vitro</i>. Inhibition of <i>S. aureus</i> growth <i>in vivo</i> was tested by pre-inoculating mouse mammary glands with <i>S. simulans</i> 24 hours prior to <i>S. aureus</i> inoculation and harvesting mammary gland tissue 24 hours after <i>S. aureus</i> superinfection. Mammary glands were processed for bacterial load quantification, cytokine profiling, and histological processing. Pre-inoculation of either <i>S. simulans</i> strain resulted in reduced <i>S. aureus</i> load in mammary tissue. Superinfection of <i>S. simulans</i> and <i>S. aureus</i> showed no difference in inflammation severity compared to <i>S. aureus</i> alone, although <i>S. simulans</i> pre-inoculation significantly increased expression of IL-10. These results demonstrate that protection against <i>S. aureus</i> mastitis is possible by prior colonization of mammary tissue by <i>S. simulans</i> independent of <i>in vitro</i> growth inhibition capacity. This work will foster future research aiming to fully understand the variety of roles NAS strains play in bovine mastitis, aiding in the development toward alternative mastitis prophylaxis.IMPORTANCEBovine mastitis is a leading economic concern for dairy production globally, representing the largest reason for antimicrobial use in dairy cattle. Non-<i>aureus</i> staphylococci (NAS) are among the most frequently isolated bacteria from mild, sometimes self-limiting, intramammary infections in cattle and may be associated with a lower risk of infection by major clinical mastitis pathogens such as <i>Staphylococcus aureus</i>. This study investigated the inhibition of <i>S. aureus</i> mastitis by two NAS strains using an <i>in vivo</i> mouse mastitis model. This study demonstrated that when mammary glands are colonized by either one of these NAS strains, the ability of <i>S. aureus</i> to establish within the mouse mammary glands is reduced. These results demonstrate the long-term potential for NAS strains to become an alternative prophylactic treatment for bovine mastitis and support efforts to reduce antimicrobial dependencies in food production.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0067825"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442402/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144774572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tracing of streptococcal strains from infant stools across human body sites links site-specific prevalence to adhesins.","authors":"Ida Ormaasen, Morten Kjos, Melanie Rae Simpson, Torbjørn Øien, Lars Snipen, Knut Rudi","doi":"10.1128/aem.00196-25","DOIUrl":"10.1128/aem.00196-25","url":null,"abstract":"<p><p>Streptococci colonize various human body sites, both as commensals and as pathogens. They are early gut colonizers, but we lack strain-level information about the origin and persistence of streptococci in the non-diseased gut. To gain a greater insight into commensal streptococci in the infant gut and their presence in other body habitats, we analyzed samples from mother-infant pairs collected from multiple body sites (stool, oral cavity, vagina, breast milk). We performed whole-metagenome sequencing and isolated streptococci from 100 infant stool samples (collected at 10 days of age). To trace the streptococci at the strain level, we designed selective quantitative PCR (qPCR) primers for seven streptococcal strains, which were then used to screen the corresponding samples from the other body sites of the infants and their mothers. We found that two of the strains investigated, one <i>Streptococcus parasanguinis</i> strain and one <i>Streptococcus vestibularis</i> strain, were highly prevalent in stool samples obtained from infants and their mothers. Interestingly, the screening revealed that another <i>S. parasanguinis</i> strain, closely related to the stool-prevalent strain, displayed a completely different prevalence pattern, being most prevalent in vaginal swabs, breast milk, and oral swabs. A genotypic comparison of these two <i>S</i>. <i>parasanguinis</i> strains revealed major differences in genes encoding adhesins, suggesting that host surface attachment could be a key factor for the observed differences in body site specificity. Together, our extensive tracing of streptococci across the body sites of 100 infants and their mothers provides strain-level insight into patterns of distribution and identifies streptococcal strains prevalent in stool.IMPORTANCEStreptococci thrive on mucosal surfaces and colonize multiple human body sites, including the gut. To understand how streptococci colonize and spread between body site habitats, strain-level information about their prevalence is required; however, such knowledge is currently lacking. In this study, we isolate streptococci and perform metagenome sequencing and quantitative PCR (qPCR) on samples from a large cohort of mother-infant pairs to trace streptococcal strains in different habitats. We demonstrate how different strains prefer specific habitats. For example, we show that two closely related strains, both isolated from stool, are distributed differently across the human body, with one of them prevalent in stool samples and the other more prevalent in other samples. These results emphasize the necessity of strain-level analysis for the identification of true colonizers of a habitat.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0019625"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144815599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pathway crosstalk enables degradation of aromatic compounds in marine <i>Roseobacter</i> clade bacteria.","authors":"Huan-Wei Xu, Xiao-Yan Wang, Ying Wei, Yiqi Cao, Shu-Guang Wang, Peng-Fei Xia","doi":"10.1128/aem.00978-25","DOIUrl":"10.1128/aem.00978-25","url":null,"abstract":"<p><p>Aromatic compounds are essential raw materials for almost all sectors of human societies but also persistent environmental pollutants recalcitrant to biodegradation. The ocean serves as a significant sink for these compounds, while their biological conversion routes remain poorly understood, hindering a comprehensive understanding of the marine carbon cycle and advancements in bioremediation and biological carbon upcycling. Here, we report the degradation pathway of aromatic molecules in the marine <i>Roseobacter</i> clade bacteria through multi-omics analysis and CRISPR-Cas-based genome editing. Using <i>Roseovarius nubinhibens</i> and 4-hydroxybenzoate (4HB) as representatives, we identified the transport of 4HB via TRAP, ABC, and MFS transporters. Then, we deciphered the integral β-ketoadipate pathway responsible for aromatic degradation. Next, we discovered a distinct pathway crosstalk at the final thiolation step, which serves as an intersection node of different pathways catalyzed by the 3-oxoadipyl-CoA thiolase from the β-ketoadipate pathway and the acetyl-CoA C-acetyltransferase and acetyl-CoA C-acyltransferase from the β-oxidation pathway. Finally, we proposed <i>R. nubinhibens</i> as a novel marine platform for systems-level interrogation and bioprospecting. Our study provides a foundation for leveraging <i>Roseobacter</i> clade bacteria as novel chassis for environmental and industrial innovations.IMPORTANCEAromatic compounds lie in an essential node of carbon cycling in both natural and engineered systems. Marine bacteria orchestrate the cycling of aromatic compounds in the ocean and, as emerging chassis, have shown unusual potentials in the degradation and valorization of aromatics. However, the corresponding metabolic pathway in marine bacteria remains poorly interpreted over decades, hindering further scientific interrogation and engineering practices. Here, we deciphered the complete degradation pathway of aromatic compounds in the marine <i>Roseobacter</i> clade bacteria and established a marine platform for systems and synthetic biology. Our study provides a paradigm for biological interrogation with combined multi-omics and the cutting-edge CRISPR-Cas approaches, laying a foundation for biological innovations with marine bacteria.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0097825"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442348/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144820416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel regulatory mechanism of choline-O-sulfate and choline catabolism by two BetIs in Alphaproteobacteria.","authors":"Jia-Rong Liu, Zhen-Kun Li, Ming-Chen Wang, Na Wang, Zhi-Qing Wang, Fei-Fei Li, Yin Chen, Yu-Zhong Zhang, Hui-Hui Fu","doi":"10.1128/aem.00333-25","DOIUrl":"10.1128/aem.00333-25","url":null,"abstract":"<p><p>Choline-O-sulfate (COS) and choline are ubiquitous in the environment, and diverse bacteria catabolize them into glycine betaine for osmoprotection or as a carbon and/or nitrogen source. The characterized <i>bet</i> genes involved in COS and choline catabolism are usually clustered in the genome with one regulatory gene, <i>betI</i>. Here, we report a novel regulatory mechanism of COS and choline catabolism by two BetIs in the model marine Roseobacter group bacterium <i>Ruegeria pomeroyi</i> DSS-3. The insertion of two unrelated genes divided the <i>R. pomeroyi</i> DSS-3 <i>bet</i> cluster into two parts, with each part having its own regulatory <i>betI</i>. BetI1 deregulates the transcription of the <i>betI1-betC</i> operon and <i>betB</i> in the presence of choline. COS and choline induce the transcription of the structural genes while repressing the regulatory gene of the <i>betI2-betTA</i> divergon. Two palindromes with one shared flanking sequence in the intergenic fragment of this divergon are recognized by BetI2. The affinities of BetI2 to these two <i>betI2 boxes</i> are fine-tuned by the binding of the effector choline. Bioinformatic analysis indicated that two <i>betIs</i> exist widely in members of Alphaproteobacteria. This study elucidates a novel regulatory pattern of COS and choline catabolism in abundant bacteria.IMPORTANCECholine and its sulfonium analog choline-O-sulfate (COS) are ubiquitous, and their catabolism by the bacterial choline-to-glycine betaine pathway generates a potent osmoprotectant, glycine betaine, and also provides carbon and nitrogen sources. In contrast to previously characterized modes executed by one regulatory BetI, in this study, we elucidate a novel regulatory mechanism of COS and choline catabolism by two BetIs in the model marine Roseobacter group bacterium <i>Ruegeria pomeroyi</i> DSS-3. The two BetIs control distinct steps of COS and choline catabolism and respond differently to osmotic stress. This study indicates that the two BetIs regulatory mode is a long-overlooked mechanism adopted by abundant bacteria.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0033325"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442346/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144833792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preconditioned microbial communities in electrochemical sensing: initial assessment of detection capabilities and durability.","authors":"Yashawini Phriya Rauichandran, Kai Ling Yu, Mohd Nur Ikhmal Salehmin, Hassan Mohamed, Halimah Badioze Zaman, Samet Şahin, Eileen H Yu, Ahmad Razi Othman, Wei Lun Ang, Swee Su Lim","doi":"10.1128/aem.01151-25","DOIUrl":"10.1128/aem.01151-25","url":null,"abstract":"<p><p>This study explores the role of pre-colonized microbial cultures in enhancing the long-term effectiveness of microbial electrochemical sensors for water quality monitoring. Microbial electrochemical sensors rely on specific functional microorganisms to detect and signal changes in environmental water quality. Pre-colonization of these cultures on the sensor's electrode can promote sustained detection sensitivity. This study investigates how specific microbial groups-<i>Geobacter</i> species, sulfate-reducing bacteria (SRB), and acetogen microbes-affect sensor performance when initially enriched and transferred to the biosensor. In the pre-enrichment phase, microbes were incubated in three defined media: <i>Geobacter</i> medium, SRB medium, and acetogen medium. Each culture was cycled through three 48 h incubation periods to establish dominant microbial populations and then introduced to independent biosensors. A control sensor was seeded with natural inoculum from Tasek Kejuruteraan UKM. The results showed that <i>Geobacter</i>-enriched biosensors quickly generated strong electrical signals by oxidizing substrates at the anode, marking them as the most effective at facilitating electron transfer. SRB-enriched sensors produced negative signals, as SRB consumed electrons and thrived at the cathode. <i>Acetogen</i>-enriched biosensors exhibited slower, indirect electron transfer, with lower electrochemical activity. In contrast, the control sensor displayed only minimal increases in signal strength over time. The <i>Geobacter</i>-enriched biosensor, which achieved a significant current drop from 0.478 mA to 0.093 mA (an ~80.5% decrease) upon pollutant exposure, demonstrated the fastest response to rising pollutant levels, followed by SRB, acetogen, and the control. These findings emphasize the importance of starting with targeted microbial populations to optimize biosensor functionality for environmental monitoring applications.IMPORTANCEMicrobial electrochemical sensors are widely recognized as effective tools for environmental monitoring and water quality assessment. Numerous studies have explored the enrichment and adaptation of microbial communities in various environmental conditions, focusing on their interactions, survival, and metabolic performance. However, a critical gap remains largely overlooked-specifically, the importance of the biosensor start-up procedure and the selection of initial microbial populations. The presence of specific electrogenic bacteria at the sensing terminal during start-up plays a vital role in initiating and sustaining biosensor functionality. In this study, we aim to address this gap by not only examining the performance of the biosensor system itself but also emphasizing the role of pre-enriched microbial communities. Our approach focuses on building a healthy, functional, and responsive biosensing platform by optimizing microbial colonization from the onset.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0115125"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442381/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144939804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inactivation of KhpB (EloR/Jag) in <i>Lactococcus cremoris</i> increases uptake of the compatible solute glycine-betaine and enhances osmoresistance.","authors":"Yuwei Xiang, Huong Thi Pham, Yosephine Gumulya, Zhao-Xun Liang, Esteban Marcellin, Mark S Turner","doi":"10.1128/aem.00914-25","DOIUrl":"https://doi.org/10.1128/aem.00914-25","url":null,"abstract":"<p><p>The second messenger cyclic-di-AMP (c-di-AMP) is a signaling molecule widely present in gram-positive bacteria, where it regulates osmotic resistance by controlling potassium and compatible solute transport. Our previous studies using a <i>Lactococcus cremoris</i> model strain demonstrated that mutants with elevated c-di-AMP can overcome osmosensitivity through mutations enhancing potassium transporter activity. To identify additional mechanisms that enhance osmoresistance, we conducted a salt-resistance suppressor screen in an industrial <i>L. cremoris</i> strain. Using a spontaneous GdpP phosphodiesterase mutant with high c-di-AMP, we isolated salt-resistant suppressor mutants harboring six independent mutations in the <i>khpB</i> gene. These <i>khpB</i> mutants maintained elevated c-di-AMP levels comparable to the parental <i>gdpP</i> mutant. Inactivating <i>khpB</i> in wild-type and <i>gdpP</i> mutant laboratory <i>L. cremoris</i> strains similarly enhanced osmoresistance. KhpB (also known as EloR/Jag) is a putative RNA-binding protein, and its inactivation increased RNA transcript and protein expression of the glycine-betaine transporter BusAA-AB, elevating intracellular glycine-betaine uptake. Additionally, <i>khpB</i> disruption resulted in reduced cell size and enhanced secretion of native cell wall-degrading enzymes. Thus, KhpB likely acts as an indirect repressor of osmoresistance in <i>L. cremoris</i> by negatively regulating glycine-betaine transporter production.IMPORTANCE<i>Lactococcus cremoris</i> is a model lactic acid bacterium and an industrially valuable fermentation starter culture. Similar to other gram-positive bacteria, <i>L. cremoris</i> utilizes the nucleotide messenger c-di-AMP to manage responses to osmotic stress. A suppressor screen aimed at restoring salt resistance in a high c-di-AMP mutant identified several independent mutations within the <i>khpB</i> gene. Our results demonstrate that <i>khpB</i> disruption elevates intracellular glycine-betaine concentrations, a prominent osmoprotectant. Notably, <i>khpB</i> inactivation also reduced cell size and enhanced the secretion of native cell wall-degrading enzymes. This study thus reveals KhpB as a negative regulator of osmotic stress resistance in <i>L. cremoris</i>, thereby expanding our understanding of bacterial osmoadaptation mechanisms.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0091425"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145074327","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":"CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K.","authors":"Jonas Fernbach, Jasmin Baggenstos, Ellen-Aleksandra Svorjova, Jeannine Riedo, Shawna McCallin, Martin J Loessner, Samuel Kilcher","doi":"10.1128/aem.02014-24","DOIUrl":"10.1128/aem.02014-24","url":null,"abstract":"<p><p><i>Staphylococcus aureus</i> is a major opportunistic pathogen, increasingly difficult to treat due to rising resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among <i>S. aureus</i> phages, the strictly lytic, broad-host-range members of the <i>Twortvirinae</i> subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized <i>Twortvirus</i> K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (<i>nluc</i>)-encoding reporter phage (K::<i>nluc</i>) and tested as a bioluminescence-based approach for identifying viable <i>Staphylococcus</i> cells. Independent of their phage-resistance profile, 100% of tested clinical <i>S. aureus</i> isolates emitted bioluminescence upon K::<i>nluc</i> challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating <i>S. aureus</i> detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic <i>Twortvirinae</i> phages to combat drug-resistant <i>S. aureus</i> strains.IMPORTANCEPhage engineering, the process of modifying bacteriophages to enhance or customize their properties, offers significant potential for advancing precision antimicrobial therapies and diagnostics. While methods for engineering small <i>Staphylococcus</i> phage genomes are well-established, larger <i>Staphylococcus</i> phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of <i>Twortvirinae</i>, a subfamily of <i>Staphylococcus</i> phages known for their broad host range and strictly lytic lifestyle, making them highly relevant for diagnostic and therapeutic applications. Using this method, we successfully developed a phage-based diagnostic tool capable of rapid and sensitive detection of <i>S. aureus</i> cells across various matrices. This approach has the potential to extend beyond diagnostics, enabling applications such as phage-mediated delivery of antimicrobial effector proteins in the future.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0201424"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442396/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144774571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"State-of-the-art methods for quantifying microbial polyhydroxyalkanoates.","authors":"Eric M Conners, Arpita Bose","doi":"10.1128/aem.00274-25","DOIUrl":"10.1128/aem.00274-25","url":null,"abstract":"<p><p>Polyhydroxyalkanoates are a diverse class of microbially synthesized polymers that are used to make bioplastics with a wide range of applications. As interest in polyhydroxyalkanoates (PHAs) grows, researchers are faced with a challenge: how best to use the resources at their disposal to reliably quantify PHA produced by their microbe(s) of choice. Investigators must weigh the pros and cons of each method against logistical constraints (e.g., time, money, and equipment) and technical concerns (e.g., accuracy and sensitivity). At the same time, the broader community of scientists researching PHAs should aspire to land on a set of best practices. To this end, we must continually audit our methods. Here, we offer readers a snapshot of popular and emerging approaches for quantifying PHA in the lab. For each method, we provide an overview<b>,</b> list the primary equipment, briefly describe the methods, including improvements or iterations, and discuss the pros and cons of the approach. Along the way, we highlight gaps in research and make recommendations about best practices and future directions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0027425"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442350/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144783314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of degradation conditions and elucidation of novel biodegradation pathways for sulfamonomethoxine by a novel <i>Bacillus</i> strain.","authors":"Xiujuan Wang, Jingtong Li, Chunyan Chen, Zifeng Luo, Yuwan Pang, Hongxing Tu, Xiaojun Lin, Cuifen Long, Qianyi Cai, Zebin Wei, Jinrong Qiu","doi":"10.1128/aem.01329-25","DOIUrl":"10.1128/aem.01329-25","url":null,"abstract":"<p><p>As a commonly used sulfonamide antibiotic, the efficient reduction of sulfamonomethoxine (SMM) residue in the environment is a critical issue that urgently needs to be addressed. However, there is limited understanding of the microbial conditions needed for efficient SMM degradation and its mechanisms. Therefore, this study screened a new strain, <i>Bacillus</i> sp. DLY-11, from swine manure compost with significant SMM degradation capability, and utilized response surface methodology (RSM) based on Box-Behnken design to optimize the degradation conditions. The results showed that under conditions of a 5% inoculation volume, a temperature of 59.1°C, a pH value of 7.10, and 0.45 g/L MgSO₄, strain DLY-11 could degrade 98.8% of 20 mg/L SMM within 48 h. Product analysis identified six potential transformation products and proposed two potential biodegradation pathways of SMM, including C-N bond cleavage, hydroxylation, and SO₂ release. Particularly, we discovered a novel degradation pathway that has not been reported before. This study not only introduced a new strain for efficient SMM degradation but also optimized conditions and revealed new degradation pathways. These findings addressed gaps in bacterial SMM degradation pathways, offering theoretical and technical support for bioremediating antibiotic pollutants in animal husbandry.</p><p><strong>Importance: </strong>The discovery of a new <i>Bacillus</i> sp., strain DLY-11, from aerobically composted swine manure offers significant environmental benefits by efficiently degrading 98.8% of 20 mg/L sulfamonomethoxine (SMM) within 48 hours under optimal conditions (5% inoculation volume, 59.1°C, pH 7.10, 0.45 g/L MgSO4). This strain introduces a new tool for reducing SMM antibiotic pollution and reveals a novel degradation pathway, enhancing our understanding of SMM biodegradation mechanisms and supporting targeted bioremediation strategies.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0132925"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442388/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144820415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simultaneous detection of five shrimp pathogens using a single-tube EvaGreen real-time PCR assay with differential melting temperature.","authors":"Haoyu Lou, Xuan Li, Guohao Wang, Kaisong Zhang, Kejun Wang, Qiongying Tang, Guoliang Yang, Peng Jia, Jinbo Xiong, Jie Huang, Xuan Dong","doi":"10.1128/aem.00591-25","DOIUrl":"10.1128/aem.00591-25","url":null,"abstract":"<p><p>The World Organisation for Animal Health (WOAH) has assessed crustacean diseases, such as infections with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), decapod iridescent virus 1 (DIV1), and acute hepatopancreatic necrosis disease (AHPND), as listed diseases, and infection with <i>Ecytonucleospora hepatopenaei</i> (EHP) as an emerging disease, all of which significantly threaten the shrimp industry. This study developed a quintuplex EvaGreen-based melting curve real-time PCR method for the simultaneous detection of WSSV, IHHNV, DIV1, AHPND-causing <i>Vibrio</i> (<i>V</i>_AHPND), and EHP. In the specific assay, only the target pathogen demonstrated efficient and detectable amplification, thereby indicating that the method exhibits high specificity. Regarding sensitivity testing, the five pathogens were detected at a concentration of 1.0 × 10¹ copies/μL. Each concentration gradient was evaluated in triplicate, and the coefficient of variation for each gradient remained below 3.38%, thereby affirming that the method demonstrates highly repeatability. We tested the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of our method using a total of 800 clinical samples which were gathered from the shrimp farming regions in China. The newly established method in this study demonstrated a DSe above 89.74% for the five pathogens and a DSp of 100%. The quintuplex EvaGreen real-time PCR method developed here offers an accurate and efficient approach for EHP, WSSV, <i>V</i>_AHPND, IHHNV, and DIV1.IMPORTANCECrustacean diseases, such as infections with WSSV, IHHNV, DIV1, <i>V</i>_AHPND, and EHP, pose a significant threat to the global shrimp industry, leading to substantial economic losses. Rapid, accurate, and simultaneous detection of these pathogens is crucial for effective disease management and biosecurity in shrimp farming. In this study, we developed a quintuplex EvaGreen-based melting curve real-time PCR method that enables the simultaneous detection of these five major shrimp pathogens with exceptional specificity, sensitivity, and repeatability. By evaluating 800 clinical samples, our method demonstrated high diagnostic sensitivity and specificity, making it a valuable tool for early pathogen detection and disease control. This novel approach can help mitigate disease outbreaks, improve shrimp farm productivity, and support the sustainable development of the aquaculture industry.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0059125"},"PeriodicalIF":3.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12442405/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144820418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}