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Advances in research and development have led to new technologies, tools and analytical capabilities bolstering the field of microbial forensics. However, a parallel commitment to education and training in this field is needed to prepare the next generation of scientists. Education and training are needed to: Improve first responder preparation and awareness, essential to public safety and preservation of the evidence integrity; Provide a larger pool of trained microbial forensics practitioners and; Educate the public and policy makers on the capabilities and limitations of microbial forensics. This chapter provides suggestions for topics, core courses, laboratories, skills, training, education programs, curricular guidelines and resources for microbial forensics that have been codified from a variety of sources including the American Society of Microbiology and American Academy of Forensic Sciences.

Additional file 3 Table S5. Median values of abiotic river water measurements.

Additional file 1 Fig. S1. Map of study site.

Additional file 2 S1 File. Sapro assay design and validation.

In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China causing severe morbidity and mortality. Since then, the virus has swept across the globe causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, provided to us by the Arizona Department of Health Services, and at Arizona State University and the University of Arizona, collected as part of community surveillance projects. Phylogenetic analysis of 79 genomes we generated from across Arizona revealed a minimum of 9 distinct introductions throughout February and March. We show that >80% of our sequences descend from clades that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related cases in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020.

Additional file 2: Table S1. Leptospira quantity data extracted from the articles analyzed in this meta-analysis.

Additional file 4: Table S3. Estimated quantity of Leptospira shed by animals.

Additional file 3: Table S2. Urine volume excreted by animals.

Core genome phylogenies of Group I strains. A) Core genome phylogeny of C. botulinum Group I strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 182,200 core genome SNPs produced with NASP [61] using C. sporogenes ATCC 15579 [GenBank:ABKW00000000] as the reference genome. The consistency index is 0.56, and the retention index is 0.91 (computed with the R package phangorn). B) Core genome phylogeny of Group I strains inferred with FastTree2 [54] on a 1780-character core SNP matrix generated with kSNP [59]. The consistency index is 0.64, and the retention index is 0.93. The phylogenies were rooted with the clade that includes C. sporogenes and C. botulinum bont/B serotypes (bottom of Figure). Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 5 and Additional file 4: Figure S1), the overall topology of the Group I tree appears robust. The pairwise overall topological scores computed by Compare2Trees [69] range from 80 to 86 % for the phylogenies presented in Fig. 5 and (Additional file 4: Figure S1). (PDF 59 kb)

Dendrograms clustering Group I and Group II strains by BSR values. A) A dendrogram generated by clustering Group I strains by BSR values with an average linkage method in MeV [76]. B) A dendrogram generated by clustering Group II strains by BSR values with an average linkage method in MeV [76]. (PDF 41 kb)

bont gene phylogeny. A phylogeny inferred with FastTree2 [54] on a nucleotide alignment (MUSCLE [57]) of botulinum neurotoxin genes. Gray circles indicate bootstrap values over 90Â %. The tree was rooted with tetanus toxin gene sequences in FigTree [55]. (PDF 54 kb)

Core genome phylogenies of Group II strains. A) Core genome phylogeny of C. botulinum Group II strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 200,276 core genome SNPs produced with NASP [61] using C. botulinum strain Alaska E43 [GenBank:CP001078] as the reference. The consistency index is 0.81, and the retention index is 0.90 (computed with the R package phangorn). B) Core genome phylogeny of Group II C. botulinum strains inferred with FastTree2 [54] on a 35,382-character core SNP matrix generated with kSNP [59]. The consistency index is 0.83, and the retention index is 0.90. The phylogenies were rooted with the clade that includes strains Eklund 202F, KAPB-3, Eklund 17B and CDC 66177. Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 6 and Additional file 6: Figure S3), Group II strains are separated into two distinct clades. The pairwise overall topological scores computed by Compare2Trees [69] range from 93 to 100 %. (PDF 35 kb)

bont gene phylogeny. A phylogeny inferred with FastTree2 [54] on a nucleotide alignment (MUSCLE [57]) of botulinum neurotoxin genes. Gray circles indicate bootstrap values over 90Â %. The tree was rooted with tetanus toxin gene sequences in FigTree [55]. (PDF 54 kb)

Group I concatenated MLST gene phylogeny. Phylogeny of aligned (MUSCLE [57]) and concatenated MLST genes for Group I genomes inferred with FastTree2 [54]. The MLST profile included aceK, aroE, hsp60, mdh, oppB, recA and rpoB [21]. Taxa labeled ST are 83 sequence types available from PubMLST [77, 78]. Investigation of the concatenated MLST gene phylogeny suggests that diverse BoNT-producing strains have yet to be whole genome sequenced. (PDF 31 kb)

Group II concatenated MLST gene phylogeny. Phylogeny of aligned (MUSCLE [57]) and concatenated MLST genes for Group II genomes inferred with FastTree2 [54]. The MLST profile included 16S rRNA genes, atpD, guaA, gyrB, ilvD, lepA, mutL, oppB, pta, pyc, recA, rpoB, trpB and tuf [22]. Taxa labeled C. botulinum are WGS samples. Taxa labeled with single-word name include serotype E strains for which the MLST genes were sequenced by MacDonald and colleagues [22]. (PDF 37 kb)

Dendrograms clustering Group I and Group II strains by BSR values. A) A dendrogram generated by clustering Group I strains by BSR values with an average linkage method in MeV [76]. B) A dendrogram generated by clustering Group II strains by BSR values with an average linkage method in MeV [76]. (PDF 41 kb)

Group II concatenated MLST gene phylogeny. Phylogeny of aligned (MUSCLE [57]) and concatenated MLST genes for Group II genomes inferred with FastTree2 [54]. The MLST profile included 16S rRNA genes, atpD, guaA, gyrB, ilvD, lepA, mutL, oppB, pta, pyc, recA, rpoB, trpB and tuf [22]. Taxa labeled C. botulinum are WGS samples. Taxa labeled with single-word name include serotype E strains for which the MLST genes were sequenced by MacDonald and colleagues [22]. (PDF 37 kb)

Core genome phylogenies of Group I strains. A) Core genome phylogeny of C. botulinum Group I strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 182,200 core genome SNPs produced with NASP [61] using C. sporogenes ATCC 15579 [GenBank:ABKW00000000] as the reference genome. The consistency index is 0.56, and the retention index is 0.91 (computed with the R package phangorn). B) Core genome phylogeny of Group I strains inferred with FastTree2 [54] on a 1780-character core SNP matrix generated with kSNP [59]. The consistency index is 0.64, and the retention index is 0.93. The phylogenies were rooted with the clade that includes C. sporogenes and C. botulinum bont/B serotypes (bottom of Figure). Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 5 and Additional file 4: Figure S1), the overall topology of the Group I tree appears robust. The pairwise overall topological scores computed by Compare2Trees [69] range from 80 to 86 % for the phylogenies presented in Fig. 5 and (Additional file 4: Figure S1). (PDF 59 kb)

Group I concatenated MLST gene phylogeny. Phylogeny of aligned (MUSCLE [57]) and concatenated MLST genes for Group I genomes inferred with FastTree2 [54]. The MLST profile included aceK, aroE, hsp60, mdh, oppB, recA and rpoB [21]. Taxa labeled ST are 83 sequence types available from PubMLST [77, 78]. Investigation of the concatenated MLST gene phylogeny suggests that diverse BoNT-producing strains have yet to be whole genome sequenced. (PDF 31 kb)

Core genome phylogenies of Group II strains. A) Core genome phylogeny of C. botulinum Group II strains inferred with RAxML [63] using the ASC_GTRGAMMA model on an alignment of 200,276 core genome SNPs produced with NASP [61] using C. botulinum strain Alaska E43 [GenBank:CP001078] as the reference. The consistency index is 0.81, and the retention index is 0.90 (computed with the R package phangorn). B) Core genome phylogeny of Group II C. botulinum strains inferred with FastTree2 [54] on a 35,382-character core SNP matrix generated with kSNP [59]. The consistency index is 0.83, and the retention index is 0.90. The phylogenies were rooted with the clade that includes strains Eklund 202F, KAPB-3, Eklund 17B and CDC 66177. Gray circles indicate bootstrap values over 95 %. While there are small variations in the phylogenies generated with different methods (Fig. 6 and Additional file 6: Figure S3), Group II strains are separated into two distinct clades. The pairwise overall topological scores computed by Compare2Trees [69] range from 93 to 100 %. (PDF 35 kb)

Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships is essential for ultimately driving these systems towards desired outcomes, e.g., the enrichment in organisms capable of accumulating valuable resources during BWWT. A comparative integrated omic analysis including metagenomics, metatranscriptomics and metaproteomics was carried out to elucidate functional differences between seasonally distinct oleaginous mixed microbial communities (OMMCs) sampled from an anoxic BWWT tank. A computational framework for the reconstruction of community-wide metabolic networks from multi-omic data was developed. These provide an overview of the functional capabilities by incorporating gene copy, transcript and protein abundances. To identify functional genes, which have a disproportionately important role in community function, we define a high relative gene expression and a high betweenness centrality relative to node degree as gene-centric and network topological features, respectively. Genes exhibiting high expression relative to gene copy abundance include genes involved in glycerolipid metabolism, particularly triacylglycerol lipase, encoded by known lipid accumulating populations, e.g., Candidatus Microthrix parvicella. Genes with a high relative gene expression and topologically important positions in the network include genes involved in nitrogen metabolism and fatty acid biosynthesis, encoded by Nitrosomonas spp. and Rhodococcus spp. Such genes may be regarded as ‘keystone genes’ as they are likely to be encoded by keystone species. The linking of key functionalities to community members through integrated omics opens up exciting possibilities for devising prediction and control strategies for microbial communities in the future. Charting metabolic activity in diverse microbial communities could help scientists control the behavior of these communities, new research shows. Paul Wilmes and colleagues at the University of Luxembourg, alongside scientists from the United States, set out to characterize the biological function of bacterial communities—normally a daunting task due to the sheer number of species involved. To accomplish this, Wilmes and colleagues took a community-wide approach, collecting genomic, transcriptomic and proteomic data from lipid-accumulating microbial consortia in a wastewater treatment tank and assembling functional maps reflecting overall biochemical processes. The scientists were then able to identify ‘keystone’ genes that play a critical role in the community—for example, genes involved in fatty acid biosynthesis were particularly prominent. Such findings could guide strategies for manipulating microbial communities for optimized disease control or biofuel production in future.

Publisher Summary Disciplines related to microbial forensics are evolving rapidly. This evolution includes technology, analytical capabilities, and, equally as important, education and training. The scientific bases, applications, interpretations, and lessons learned by those who have been intimately involved in the early years of microbial forensics need to be documented and transferred to the next generation of scientists and decision makers so that we can protect society from potential harm resulting from bioterrorism and biocrime. The burgeoning field of microbial forensics should be accompanied by a parallel development of educational infrastructure and resources targeted at the next generation of practitioners, as well as diverse elements for the policy, research, and law enforcement communities. A microbial forensics education program can be broad, providing information encompassing all aspects of the field from science to policy, or more focused depending on its purpose and target audience. Policy makers must have a general understanding of microbial forensics results and better appreciation of their implications in order to effect sound and defensible policy decisions. Finally, an important group that informs the public and government is the news media. They are frequently the primary interface between the scientist and the public, making their observations, insights, or inaccuracies of great importance and impact. Educational efforts will better prepare such individuals to be informed and responsible and must be varied in depth and scope to match the target audience of various entities involved in microbial forensics.

BACKGROUND: Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships is essential for ultimately driving these systems towards desired outcomes, e.g., the enrichment in organisms capable of accumulating valuable resources during BWWT. METHODS: A comparative integrated omic analysis including metagenomics, metatranscriptomics and metaproteomics was carried out to elucidate functional differences between seasonally distinct oleaginous mixed microbial communities (OMMCs) sampled from an anoxic BWWT tank. A computational framework for the reconstruction of community-wide metabolic networks from multi-omic data was developed. These provide an overview of the functional capabilities by incorporating gene copy, transcript and protein abundances. To identify functional genes, which have a disproportionately important role in community function, we define a high relative gene expression and a high betweenness centrality relative to node degree as gene-centric and network topological features, respectively. RESULTS: Genes exhibiting high expression relative to gene copy abundance include genes involved in glycerolipid metabolism, particularly triacylglycerol lipase, encoded by known lipid accumulating populations, e.g., Candidatus Microthrix parvicella. Genes with a high relative gene expression and topologically important positions in the network include genes involved in nitrogen metabolism and fatty acid biosynthesis, encoded by Nitrosomonas spp. and Rhodococcus spp. Such genes may be regarded as ‘keystone genes’ as they are likely to be encoded by keystone species. CONCLUSION: The linking of key functionalities to community members through integrated omics opens up exciting possibilities for devising prediction and control strategies for microbial communities in the future.

Chronic rhinosinusitis (CRS) is a highly prevalent and heterogeneous condition frequently treated with antibiotics and corticosteroid therapy. However, the effect of medical therapy on sinus microbiota remains unknown.We enrolled CRS patients (n = 6) with patent maxillary antrostomies and active mucosal inflammation, who had not received antibiotics or corticosteroids in the previous 8 weeks. A pretreatment and posttreatment maxillary sinus swab was collected, from which DNA was extracted, pyrosequenced, and analyzed using a naïve Bayesian classifier and ecological analyses.Four patients showed significant improvement in endoscopic appearance. The shifts in microbiota in response to therapy were highly individualized. There was no single common microbiota profile among patients with similar clinical outcomes, but overall there was significant decrease in microbiota diversity (t(5) = 2.05, p = 0.10) and evenness (t(5) = 2.28, p = 0.07) after treatment.Our findings strongly correlate with earlier studies that examined the impact of antibiotics on human microbiota. We observed that posttreatment, patients frequently became colonized by taxa that are less susceptible to the prescribed antibiotics. Our findings highlight the challenge in seeking generalizable diagnostic and therapeutic options in CRS, particularly regarding microbiological response and outcomes.

Teak tree (Tectona grandis L.f.) is a tropical tree specie naturally distributed in Southeast Asia, where it is also widely planted. It is planted as exotic specie in Africa, South and Central America. Tree improvement activities have been initiated in several countries including Costa Rica, which it has become the largest teak developer in Latin America. Teak sprouts were collected in a 4-yearold progeny test from the best two and the worst two families for volume as well as for stem quality traits. A DNA extraction protocol described before was modified in order to yield high quality DNA.AFLP reactions were performed as described previously. Seven selective primer combinations (EACG + M-CCG, E-ACT +M-CCG, E-AGC + M-CCG, E-ACG + M-CTC, E-ACT + M-CTC, E-AGC + M-CTC, E-AGG + M-CCA) resulted in 330 scoreable markers. A large number of markers were obtained when families where analyzed separately from its performance. Genetic distances based on AFLP markers frequencies were used to construct a dendrogram among families. The best quality performer families grouped widely separated from the worst performers, sharing 61.63% of their genetic elements. In volume, worst performer families grouped sharing 45.85% of their genetic elements.

Derek S Sarovich,1,2,* Erin P Price,1,2,* Direk Limmathurotsakul,3 James M Cook,1 Alex T Von Schulze,1 Spenser R Wolken,1 Paul Keim,1 Sharon J Peacock,3,4 Talima Pearson1 1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA; 2Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Darwin, Australia; 3Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 4Department of Medicine, University of Cambridge, Cambridge, United Kingdom*These authors contributed equally to this workAbstract: Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.Keywords: Burkholderia pseudomallei, ceftazidime, antibiotic resistance, melioidosis, β-lactamase, penA

Melioidosis is an emerging infectious disease of humans and animals in the tropics caused by the soil bacterium Burkholderia pseudomallei. Despite high fatality rates, the ecology of B.pseudomallei remains unclear. We used a combination of field and laboratory studies to investigate B.pseudomallei colonization of native and exotic grasses in northern Australia. Multivariable and spatial analyses were performed to determine significant predictors for B.pseudomallei occurrence in plants and soil collected longitudinally from field sites. In plant inoculation experiments, the impact of B.pseudomallei upon these grasses was studied and the bacterial load semi-quantified. Fluorescence in situ hybridization and confocal laser scanning microscopy were performed to localize the bacteria in plants. Burkholderia pseudomallei was found to inhabit not only the rhizosphere and roots but also aerial parts of specific grasses. This raises questions about the potential spread of B.pseudomallei by grazing animals whose droppings were found to be positive for these bacteria. In particular, B.pseudomallei readily colonized exotic grasses introduced to Australia for pasture. The ongoing spread of these introduced grasses creates new habitats suitable for B.pseudomallei survival and may be an important factor in the evolving epidemiology of melioidosis seen both in northern Australia and elsewhere globally.

This paper discusses an efficient approach to statically compute a WCET that is "soft" rather than "hard". The goal of most timing analysis is to determine a guaranteed WCET; however this execution time may be far above the actual distribution of observed execution times. A WCET estimate that bounds the execution time 99% of the time may be more useful for a designer in a soft real-time environment. This paper discusses an approach to measure the execution time distribution by a hardware simulator, and a path-based timing analysis approach to derive a static estimation of this same distribution. The technique can find a soft WCET for loops having any number of paths.

The risk and threat of bioterrorism and biocrime have become a large concern and challenge for governments and society to enhance biosecurity. Law enforcement plays an important role in assessing and investigating activities involved in an event of bioterrorism or biocrime. Key to a successful biosecurity program is increased awareness and early detection of threats facilitated by an integrated network of responsibilities and capabilities from government, academic, private, and public assets. To support an investigation, microbial forensic sciences are employed to analyze and characterize forensic evidence with the goal of attribution or crime scene reconstruction. Two different molecular biology-based assays – real time polymerase chain reaction (PCR) and repetitive element PCR – are described and demonstrate how molecular biology tools may be utilized to aid in the investigative process. Technologies relied on by microbial forensic scientists need to be properly validated so that the methods used are understood and so that interpretation of results is carried out within the limitations of the assays. The three types of validation are preliminary, developmental, and internal. The first is necessary for rapid response when a threat is imminent or an attack has recently occurred. The latter two apply to implementation of routinely used procedures.

The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China causing severe morbidity and mortality. Since then, the virus has swept across the globe causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, provided to us by the Arizona Department of Health Services, and at Arizona State University and the University of Arizona, collected as part of community surveillance projects. Phylogenetic analysis of 79 genomes we generated from across Arizona revealed a minimum of 9 distinct introductions throughout February and March. We show that >80% of our sequences descend from clades that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related cases in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020., medRxiv

Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs.

Table S1. Sequencing quality and coverage. Quality breadth is based on the total reference size, 2,780,693 bases, after duplicated regions were removed. In order to pass quality filtering, the loci had to have >10× coverage and a SNP state had to be in >90 % of the reads to be considered. Mean coverage was calculated using a genome size of 2.8 Mb. (DOC 54 kb)

Table S2. Multi-Locus Sequence Type results obtained from WGS data using SRST2. NF indicates not found and (*) indicates mismatches. (DOC 52Â kb)

Table S2. Multi-Locus Sequence Type results obtained from WGS data using SRST2. NF indicates not found and (*) indicates mismatches. (DOC 52Â kb)

Table S1. Sequencing quality and coverage. Quality breadth is based on the total reference size, 2,780,693 bases, after duplicated regions were removed. In order to pass quality filtering, the loci had to have >10× coverage and a SNP state had to be in >90 % of the reads to be considered. Mean coverage was calculated using a genome size of 2.8 Mb. (DOC 54 kb)

With the likelihood that changes in global climate will adversely affect the soil C reservoir in the northern circumpolar permafrost zone, an understanding of the potential role of diazotrophic communities in enhancing biological N2 fixation, which constrains both plant production and microbial decomposition in tundra soils, is important in elucidating the responses of soil microbial communities to global climate change. A recent study showed that the composition of the diazotrophic community in a tundra soil exhibited no change under a short-term (1.5-year) winter warming experiment. However, it remains crucial to examine whether the lack of diazotrophic community responses to warming is persistent over a longer time period as a possibly important mechanism in stabilizing tundra soil C. Through a detailed characterization of the effects of winter warming on diazotrophic communities, we showed that a long-term (5-year) winter warming substantially enhanced diazotrophic abundance and altered community composition, though soil depth had a stronger influence on diazotrophic community composition than warming. These changes were best explained by changes in soil moisture, soil thaw duration, and plant biomass. These results provide crucial insights into the potential factors that may impact future C and N availability in tundra regions., Tundra ecosystems are typically carbon (C) rich but nitrogen (N) limited. Since biological N2 fixation is the major source of biologically available N, the soil N2-fixing (i.e., diazotrophic) community serves as an essential N supplier to the tundra ecosystem. Recent climate warming has induced deeper permafrost thaw and adversely affected C sequestration, which is modulated by N availability. Therefore, it is crucial to examine the responses of diazotrophic communities to warming across the depths of tundra soils. Herein, we carried out one of the deepest sequencing efforts of nitrogenase gene (nifH) to investigate how 5 years of experimental winter warming affects Alaskan soil diazotrophic community composition and abundance spanning both the organic and mineral layers. Although soil depth had a stronger influence on diazotrophic community composition than warming, warming significantly (P