Host-associated microbiomes drive structure and function of marine ecosystems.

The significance of symbioses between eukaryotic hosts and microbes extends from the organismal to the ecosystem level and underpins the health of Earth's most threatened marine ecosystems. Despite rapid growth in research on host-associated microbes, from individual microbial symbionts to host-associated consortia of significantly relevant taxa, little is known about their interactions with the vast majority of marine host species. We outline research priorities to strengthen our current knowledge of host-microbiome interactions and how they shape marine ecosystems. We argue that such advances in research will help predict responses of species, communities, and ecosystems to stressors driven by human activity and inform future management strategies.

Gut Check: The evolution of an educational board game.

The "gamification" of science has gained a lot of traction in recent years, and games that convey scientific concepts or themes are increasingly popular. While a number of existing games touch on microbiology, very few consider the beneficial (as opposed to the detrimental) aspects of microbes. We designed a board game called "Gut Check: The Microbiome Game" to fill this gap. The game is meant to be both educational as well as challenging and fun. Here we discuss the development of the game, some of the logistics of game development in this context, and offer suggestions for others thinking of similar projects.

Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016.

During the water crisis in Flint, Michigan, USA (2014-2015), 2 outbreaks of Legionnaires' disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2-1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Characterization of the microbiome and volatile compounds in anal gland secretions from domestic cats (Felis catus) using metagenomics and metabolomics.

Many mammals rely on volatile organic chemical compounds (VOCs) produced by bacteria for their communication and behavior, though little is known about the exact molecular mechanisms or bacterial species that are responsible. We used metagenomic sequencing, mass-spectrometry based metabolomics, and culturing to profile the microbial and volatile chemical constituents of anal gland secretions in twenty-three domestic cats (Felis catus), in attempts to identify organisms potentially involved in host odor production. We found that the anal gland microbiome was dominated by bacteria in the genera Corynebacterium, Bacteroides, Proteus, Lactobacillus, and Streptococcus, and showed striking variation among individual cats. Microbiome profiles also varied with host age and obesity. Metabolites such as fatty-acids, ketones, aldehydes and alcohols were detected in glandular secretions. Overall, microbiome and metabolome profiles were modestly correlated (r = 0.17), indicating that a relationship exists between the bacteria in the gland and the metabolites produced in the gland. Functional analyses revealed the presence of genes predicted to code for enzymes involved in VOC metabolism such as dehydrogenases, reductases, and decarboxylases. From metagenomic data, we generated 85 high-quality metagenome assembled genomes (MAGs). Of importance were four MAGs classified as Corynebacterium frankenforstense, Proteus mirabilis, Lactobacillus johnsonii, and Bacteroides fragilis. They represent strong candidates for further investigation of the mechanisms of volatile synthesis and scent production in the mammalian anal gland.

The challenge of SARS-CoV-2 environmental monitoring in schools using floors and portable HEPA filtration units: Fresh or relic RNA?

Testing surfaces in school classrooms for the presence of SARS-CoV-2, the virus that causes COVID-19, can provide public-health information that complements clinical testing. We monitored the presence of SARS-CoV-2 RNA in five schools (96 classrooms) in Davis, California (USA) by collecting weekly surface-swab samples from classroom floors and/or portable high-efficiency particulate air (HEPA) units (n = 2,341 swabs). Twenty-two surfaces tested positive, with qPCR cycle threshold (Ct) values ranging from 36.07-38.01. Intermittent repeated positives in a single room were observed for both floor and HEPA filter samples for up to 52 days, even following regular cleaning and HEPA filter replacement after a positive result. We compared the two environmental sampling strategies by testing one floor and two HEPA filter samples in 57 classrooms at Schools D and E. HEPA filter sampling yielded 3.02% and 0.41% positivity rates per filter sample collected for Schools D and E, respectively, while floor sampling yielded 0.48% and 0% positivity rates. Our results indicate that HEPA filter swabs are more sensitive than floor swabs at detecting SARS-CoV-2 RNA in interior spaces. During the study, all schools were offered weekly free COVID-19 clinical testing through Healthy Davis Together (HDT). HDT also offered on-site clinical testing in Schools D and E, and upticks in testing participation were observed following a confirmed positive environmental sample. However, no confirmed COVID-19 cases were identified among students associated with classrooms yielding positive environmental samples. The positive samples detected in this study appeared to contain relic viral RNA from individuals infected before the monitoring program started and/or RNA transported into classrooms via fomites. High-Ct positive results from environmental swabs detected in the absence of known active infections supports this conclusion. Additional research is needed to differentiate between fresh and relic SARS-CoV-2 RNA in environmental samples and to determine what types of results should trigger interventions.

SARS-CoV-2 RNA Is Readily Detectable at Least 8 Months after Shedding in an Isolation Facility.

Environmental monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for research and public health purposes has grown exponentially throughout the coronavirus disease 2019 (COVID-19) pandemic. Monitoring wastewater for SARS-CoV-2 provides early warning signals of virus spread and information on trends in infections at a community scale. Indoor environmental monitoring (e.g., swabbing of surfaces and air filters) to identify potential outbreaks is less common, and the evidence for its utility is mixed. A significant challenge with surface and air filter monitoring in this context is the concern of "relic RNA," noninfectious RNA found in the environment that is not from recently deposited virus. Here, we report detection of SARS-CoV-2 RNA on surfaces in an isolation unit (a university dorm room) for up to 8 months after a COVID-19-positive individual vacated the space. Comparison of sequencing results from the same location over two time points indicated the presence of the entire viral genome, and sequence similarity confirmed a single source of the virus. Our findings highlight the need to develop approaches that account for relic RNA in environmental monitoring. IMPORTANCE Environmental monitoring of SARS-CoV-2 is rapidly becoming a key tool in infectious disease research and public health surveillance. Such monitoring offers a complementary and sometimes novel perspective on population-level incidence dynamics relative to that of clinical studies by potentially allowing earlier, broader, more affordable, less biased, and less invasive identification. Environmental monitoring can assist public health officials and others when deploying resources to areas of need and provides information on changes in the pandemic over time. Environmental surveillance of the genetic material of infectious agents (RNA and DNA) in wastewater became widely applied during the COVID-19 pandemic. There has been less research on other types of environmental samples, such as surfaces, which could be used to indicate that someone in a particular space was shedding virus. One challenge with surface surveillance is that the noninfectious genetic material from a pathogen (e.g., RNA from SARS-CoV-2) may be detected in the environment long after an infected individual has left the space. This study aimed to determine how long SARS-CoV-2 RNA could be detected in a room after a COVID-positive person had been housed there.

Viral Load Among Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Persons Infected With the SARS-CoV-2 Delta Variant.

We found no significant difference in cycle threshold values between vaccinated and unvaccinated persons infected with severe acute respiratory syndrome coronavirus 2 Delta, overall or stratified by symptoms. Given the substantial proportion of asymptomatic vaccine breakthrough cases with high viral levels, interventions, including masking and testing, should be considered in settings with elevated coronavirus disease 2019 transmission.

Air versus Water Chilling of Chicken: a Pilot Study of Quality, Shelf-Life, Microbial Ecology, and Economics.

The United States' large-scale poultry meat industry is energy and water intensive, and opportunities may exist to improve sustainability during the broiler chilling process. By USDA regulation, after harvest the internal temperature of the chicken must be reduced to 40°F or less within 16 h to inhibit bacterial growth that would otherwise compromise the safety of the product. This step is accomplished most commonly by water immersion chilling in the United States, while air chilling methods dominate other global markets. A comprehensive understanding of the differences between these chilling methods is lacking. Therefore, we assessed the meat quality, shelf-life, microbial ecology, and techno-economic impacts of chilling methods on chicken broilers in a university meat laboratory setting. We discovered that air chilling methods resulted in superior chicken odor and shelf-life, especially prior to 14 days of dark storage. Moreover, we demonstrated that air chilling resulted in a more diverse microbiome that we hypothesize may delay the dominance of the spoilage organism Pseudomonas Finally, a techno-economic analysis highlighted potential economic advantages to air chilling compared to water chilling in facility locations where water costs are a more significant factor than energy costs.IMPORTANCE As the poultry industry works to become more sustainable and to reduce the volume of food waste, it is critical to consider points in the processing system that can be altered to make the process more efficient. In this study, we demonstrate that the method used during chilling (air versus water chilling) influences the final product microbial community, quality, and physiochemistry. Notably, the use of air chilling appears to delay the bloom of Pseudomonas spp. that are the primary spoilers in packaged meat products. By using air chilling to reduce carcass temperatures instead of water chilling, producers may extend the time until spoilage of the products and, depending on the cost of water in the area, may have economic and sustainability advantages. As a next step, a similar experiment should be done in an industrial setting to confirm these results generated in a small-scale university lab facility.

SARS-CoV-2 detection and genomic sequencing from hospital surface samples collected at UC Davis.

RATIONALE: There is little doubt that aerosols play a major role in the transmission of SARS-CoV-2. The significance of the presence and infectivity of this virus on environmental surfaces, especially in a hospital setting, remains less clear. OBJECTIVES: We aimed to analyze surface swabs for SARS-CoV-2 RNA and infectivity, and to determine their suitability for sequence analysis. METHODS: Samples were collected during two waves of COVID-19 at the University of California, Davis Medical Center, in COVID-19 patient serving and staff congregation areas. qRT-PCR positive samples were investigated in Vero cell cultures for cytopathic effects and phylogenetically assessed by whole genome sequencing. MEASUREMENTS AND MAIN RESULTS: Improved cleaning and patient management practices between April and August 2020 were associated with a substantial reduction of SARS-CoV-2 qRT-PCR positivity (from 11% to 2%) in hospital surface samples. Even though we recovered near-complete genome sequences in some, none of the positive samples (11 of 224 total) caused cytopathic effects in cultured cells suggesting this nucleic acid was either not associated with intact virions, or they were present in insufficient numbers for infectivity. Phylogenetic analysis suggested that the SARS-CoV-2 genomes of the positive samples were derived from hospitalized patients. Genomic sequences isolated from qRT-PCR negative samples indicate a superior sensitivity of viral detection by sequencing. CONCLUSIONS: This study confirms the low likelihood that SARS-CoV-2 contamination on hospital surfaces contains infectious virus, disputing the importance of fomites in COVID-19 transmission. Ours is the first report on recovering near-complete SARS-CoV-2 genome sequences directly from environmental surface swabs.

The role of fimV and the importance of its tandem repeat copy number in twitching motility, pigment production, and morphology in Legionella pneumophila.

Twitching motility, a flagella-independent type of translocation of bacteria over moist surfaces, requires an array of proteins, including FimV. To investigate the role of this protein in twitching motility in Legionella pneumophila, we have generated a knockout mutant of fimV and characterized its phenotypic effects. In addition to a major reduction in twitching motility, deletion of the fimV gene caused a number of other phenotypic effects including decreased protective pigment formation, and it also affected cell morphology. Since fimV contains a variable number of tandem repeats, which can vary according to the origin of a given strain, we have examined the importance of this variability found within the coding region of this gene. By complementing the knockout strain with constructs containing a different number of this tandem repeat, we have been able to also show that repeat copy number is important in the functioning of this gene.

Draft Genome Analysis of Christensenella minuta DSM 22607, exhibiting an unusual expansion of transporter homologs of unknown function.

Christensenella minuta was first formally described in 2012 as a member of a novel species, genus, and proposed family of Christensenellaceae. C. minuta was later shown in one study to be part of the most heritable taxonomic group in the human gut microbiome and to be enriched in people with low body mass index (BMI). Mouse work demonstrated that injection of cultured C. minuta into germ-free mice prevented the onset of obesity after a fecal transplant to the mice from high BMI individuals. Here we describe the genome sequence of C. minuta DSM 22607. Examination and analysis of the annotation revealed an unusually high number of genes predicted to be involved in carbohydrate metabolism, many of which were multiple homologs of RbsA, RbsB and RbsC, which together make up the Ribose ABC Transport System. These genes may be also involved in quorum sensing which could potentially relate to the importance of C. minuta in the gut microbiome.

Isolation and sequence-based characterization of a koala symbiont: Lonepinella koalarum.

Koalas (Phascolarctos cinereus) are highly specialized herbivorous marsupials that feed almost exclusively on Eucalyptus leaves, which are known to contain varying concentrations of many different toxic chemical compounds. The literature suggests that Lonepinella koalarum, a bacterium in the Pasteurellaceae family, can break down some of these toxic chemical compounds. Furthermore, in a previous study, we identified L. koalarum as the most predictive taxon of koala survival during antibiotic treatment. Therefore, we believe that this bacterium may be important for koala health. Here, we isolated a strain of L. koalarum from a healthy koala female and sequenced its genome using a combination of short-read and long-read sequencing. We placed the genome assembly into a phylogenetic tree based on 120 genome markers using the Genome Taxonomy Database (GTDB), which currently does not include any L. koalarum assemblies. Our genome assembly fell in the middle of a group of Haemophilus, Pasteurella and Basfia species. According to average nucleotide identity and a 16S rRNA gene tree, the closest relative of our isolate is L. koalarum strain Y17189. Then, we annotated the gene sequences and compared them to 55 closely related, publicly available genomes. Several genes that are known to be involved in carbohydrate metabolism could exclusively be found in L. koalarum relative to the other taxa in the pangenome, including glycoside hydrolase families GH2, GH31, GH32, GH43 and GH77. Among the predicted genes of L. koalarum were 79 candidates putatively involved in the degradation of plant secondary metabolites. Additionally, several genes coding for amino acid variants were found that had been shown to confer antibiotic resistance in other bacterial species against pulvomycin, beta-lactam antibiotics and the antibiotic efflux pump KpnH. In summary, this genetic characterization allows us to build hypotheses to explore the potentially beneficial role that L. koalarum might play in the koala intestinal microbiome. Characterizing and understanding beneficial symbionts at the whole genome level is important for the development of anti- and probiotic treatments for koalas, a highly threatened species due to habitat loss, wildfires, and high prevalence of Chlamydia infections.

2019 Novel Coronavirus (COVID-19) Pandemic: Built Environment Considerations To Reduce Transmission.

With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in coronavirus disease 2019 (COVID-19), corporate entities, federal, state, county, and city governments, universities, school districts, places of worship, prisons, health care facilities, assisted living organizations, daycares, homeowners, and other building owners and occupants have an opportunity to reduce the potential for transmission through built environment (BE)-mediated pathways. Over the last decade, substantial research into the presence, abundance, diversity, function, and transmission of microbes in the BE has taken place and revealed common pathogen exchange pathways and mechanisms. In this paper, we synthesize this microbiology of the BE research and the known information about SARS-CoV-2 to provide actionable and achievable guidance to BE decision makers, building operators, and all indoor occupants attempting to minimize infectious disease transmission through environmentally mediated pathways. We believe this information is useful to corporate and public administrators and individuals responsible for building operations and environmental services in their decision-making process about the degree and duration of social-distancing measures during viral epidemics and pandemics.

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus.

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.

Bacterial communities associated with cell phones and shoes.

BACKGROUND: Every human being carries with them a collection of microbes, a collection that is likely both unique to that person, but also dynamic as a result of significant flux with the surrounding environment. The interaction of the human microbiome (i.e., the microbes that are found directly in contact with a person in places such as the gut, mouth, and skin) and the microbiome of accessory objects (e.g., shoes, clothing, phones, jewelry) is of potential interest to both epidemiology and the developing field of microbial forensics. Therefore, the microbiome of personal accessories are of interest because they serve as both a microbial source and sink for an individual, they may provide information about the microbial exposure experienced by an individual, and they can be sampled non-invasively. FINDINGS: We report here a large-scale study of the microbiome found on cell phones and shoes. Cell phones serve as a potential source and sink for skin and oral microbiome, while shoes can act as sampling devices for microbial environmental experience. Using 16S rRNA gene sequencing, we characterized the microbiome of thousands of paired sets of cell phones and shoes from individuals at sporting events, museums, and other venues around the United States. CONCLUSIONS: We place this data in the context of previous studies and demonstrate that the microbiome of phones and shoes are different. This difference is driven largely by the presence of "environmental" taxa (taxa from groups that tend to be found in places like soil) on shoes and human-associated taxa (taxa from groups that are abundant in the human microbiome) on phones. This large dataset also contains many novel taxa, highlighting the fact that much of microbial diversity remains uncharacterized, even on commonplace objects.

Draft Genome Sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, Bacillus sp. Strain V59.32b, Bacillus sp. Strain MER_TA_151, and Paenibacillus sp. Strain MER_111, Isolated from Cleanrooms Where the Viking and Mars Exploration Rover Spacecraft Were Assembled.

We report the draft genome sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, and Bacillus sp. strain V59.32b, isolated from the Viking spacecraft assembly cleanroom, and Bacillus sp. strain MER_TA_151 and Paenibacillus sp. strain MER_111, isolated from the Mars Exploration Rover (MER) assembly cleanroom.

Network analysis to evaluate the impact of research funding on research community consolidation.

In 2004, the Alfred P. Sloan Foundation launched a new program focused on incubating a new field, "Microbiology of the Built Environment" (MoBE). By the end of 2017, the program had supported the publication of hundreds of scholarly works, but it was unclear to what extent it had stimulated the development of a new research community. We identified 307 works funded by the MoBE program, as well as a comparison set of 698 authors who published in the same journals during the same period of time but were not part of the Sloan Foundation-funded collaboration. Our analysis of collaboration networks for both groups of authors suggests that the Sloan Foundation's program resulted in a more consolidated community of researchers, specifically in terms of number of components, diameter, density, and transitivity of the coauthor networks. In addition to highlighting the success of this particular program, our method could be applied to other fields to examine the impact of funding programs and other large-scale initiatives on the formation of research communities.

Bacteria isolated from Bengal cat (Felis catus × Prionailurus bengalensis) anal sac secretions produce volatile compounds potentially associated with animal signaling.

In social animals, scent secretions and marking behaviors play critical roles in communication, including intraspecific signals, such as identifying individuals and group membership, as well as interspecific signaling. Anal sacs are an important odor producing organ found across the carnivorans (species in the mammalian Order Carnivora). Secretions from the anal sac may be used as chemical signals by animals for behaviors ranging from defense to species recognition to signaling reproductive status. In addition, a recent study suggests that domestic cats utilize short-chain free fatty acids in anal sac secretions for individual recognition. The fermentation hypothesis is the idea that symbiotic microorganisms living in association with animals contribute to odor profiles used in chemical communication and that variation in these chemical signals reflects variation in the microbial community. Here we examine the fermentation hypothesis by characterizing volatile organic compounds (VOC) and bacteria isolated from anal sac secretions collected from a male Bengal cat (Felis catus × Prionailurus bengalensis), a cross between the domestic cat and the leopard cat. Both left and right anal sacs of a male Bengal cat were manually expressed (emptied) and collected. Half of the material was used to culture bacteria or to extract bacterial DNA and the other half was used for VOC analysis. DNA was extracted from the anal sac secretions and used for a 16S rRNA gene PCR amplification and sequencing based characterization of the microbial community. Additionally, some of the material was plated out in order to isolate bacterial colonies. Three taxa (Bacteroides fragilis, Tessaracoccus, and Finegoldia magna) were relatively abundant in the 16S rRNA gene sequence data and also isolated by culturing. Using Solid Phase Microextraction (SPME) gas chromatography-mass spectrometry (GC-MS), we tentatively identified 52 compounds from the Bengal cat anal sac secretions and 67 compounds from cultures of the three bacterial isolates chosen for further analysis. Among 67 compounds tentatively identified from bacterial isolates, 51 were also found in the anal sac secretion. We show that the bacterial community in the anal sac consists primarily of only a few abundant taxa and that isolates of these taxa produce numerous volatiles that are found in the combined anal sac volatile profile. Several of these volatiles are found in anal sac secretions from other carnivorans, and are also associated with known bacterial biosynthesis pathways. This is consistent with the fermentation hypothesis and the possibility that the anal sac is maintained at least in part to house bacteria that produce volatiles for the host.