Animal use and care

No laboratory animals were used in this research. The owner of a male Bengal cat volunteered to have the cat’s anal sacs manually expressed by a veterinarian at the Berkeley Dog and Cat Hospital in Berkeley, CA.

Sample collection

The UC Davis Institutional Animal Care and Use Committee (IACUC) determined that no study approval was necessary for the collection of non-invasively sampled waste materials (such as anal sac secretions and feces). The client requested that the veterinarian express the anal sacs as part of a standard veterinary exam, and not for the purposes of this study.

With the owner’s consent, both left and right anal sacs were manually expressed in a male Bengal cat (F. catus × P. bengalensis) by a veterinarian at the Berkeley Dog and Cat Hospital in Berkeley, CA. Samples of anal sac secretions were collected using Puritan cotton swabs and placed in 2 mL screw cap tubes. In total seven swabs were used to collect samples: two for 16S rRNA gene PCR and sequencing, three for GC/MS analysis and two for culturing.

DNA extraction and 16S rRNA gene sequencing and analysis

Three swabs were used for 16S rRNA gene PCR and sequencing: one from the left anal sac secretion, one from the right anal sac secretion, and one unused swab (used as a control). Each of these swabs was placed into 100% ethanol prior to DNA extraction. Genomic DNA was extracted using the MoBio PowerSoil DNA Isolation kit (MoBio, Carlsbad, CA, USA). Samples were transferred to bead tubes containing C1 solution and incubated at 65 °C for 10 minutes, followed by 3 minutes of bead beating. The remaining extraction protocol was performed as directed by the manufacturer.

DNA samples were sent to the Integrated Microbiome Resource (IMR), Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University for sequencing. Bacterial diversity was characterized via PCR amplification of the 16S rRNA gene (V4-V5 region) using barcoded primers 515F and 926R [35]. PCR conditions were as follows: an initial denaturation step at 98 °C for 30 seconds, 30 cycles of 98 °C for 10 seconds, 55 °C for 30 seconds and 72 °C for 30 seconds, a final extension at 72 °C for 4 minutes 30 seconds, and a final hold at 4 °C. Prior to sequencing, the amount of input DNA per sample was normalized using a SequalPrep Normalization Plate, following the standard protocol (ThermoFisher Scientific, Wilmington, DE, USA). The final library pool was quantified using the Qubit dsDNA HS assay (Invitrogen, Carlsbad, CA). The IMR then generated 300 bp paired end sequencing reads of these PCR amplicons using v3 chemistry on an Illumina MiSeq machine.

Raw amplicon reads were demultiplexed and then processed using DADA2 v1.8, following the standard online tutorial [36]. The reads were trimmed down to 250 base pairs to remove low quality nucleotides. In addition, the quality of reads was ensured by trimming bases that did not satisfy a Q2 quality score. Reads containing Ns were discarded and we used two expected errors to filter the overall quality of the read (rather than averaging quality scores) [37]. Chimeric reads were also removed using DADA2 on a per sample basis. The remaining pairs of reads were merged into amplicon fragments and unique Amplicon Sequence Variants (ASVs) were identified. Reads that did not merge successfully were discarded. Upon completion of the DADA2 pipeline, all ASVs (n = 52) that were found in the negative control swabs were removed from the analysis, only three of these ASVs were also found in the anal sac samples. ASVs were assigned taxonomy using the DADA2 function “assignTaxonomy” and the Silva (NR v132) database [38–40]. One ASV that was assigned to “Eukaryotes” was removed. All ASVs with the same taxonomy (at the genus level) were grouped and then ranked by number of reads. No ASVs were assigned to mitochondria or chloroplast.

Bacterial culturing and identification

Anal sac secretions from two swabs from the Bengal cat were vortexed with 1 mL Phosphate Buffer Saline (PBS). Two serial 1:10 dilutions were performed and 100 μL of each dilution was plated onto Columbia Blood Agar (CBA) and Brain Heart Infusion (BHI). Plates were incubated anaerobically in a BD GasPak EZ Container System with packets of CO₂ generator for 5 days at 37 °C. Morphologically distinct colonies were streaked for isolation on both CBA and BHI. The 16S rRNA gene from each culture was PCR amplified and sequenced using Sanger sequencing using the 27F and 1391R primers. Taxonomy was assigned by the result of BLAST queries to the nr database at NCBI (excluding unnamed/environmental sequences), a species name was given in cases where the identity was >98% to only a single species.

Extraction and collection of volatiles

Cultured organisms. To extract volatiles from Bacteroides fragilis UCD-AAL1 and Tessaracoccus sp. UCD-MLA, cultures were grown in 5 mL BHI anaerobically for 24 hours at 37 °C. Three biological replicates were conducted by placing 100 μL of the culture into each of three Restek (Bellefonte, PA) tubes filled with 5 mL of BHI. Two jar blanks (no media or bacteria) and two BHI media-only blanks were used as controls. The same procedure was followed for Finegoldia magna UCD-MLG, except that cultures were grown and incubated in BHI supplemented with 5% defibrinated sheep blood (BBHI) anaerobically for 24 hours at 37 °C.

Headspace extraction was performed with Solid Phase Microextraction (SPME) fibers (Part 57912-U, Sigma Aldrich), which had 50/30 μm thickness and DVB/CAR/PDMA coating. Two SPMEs were inserted into the headspace of each Restek tube prior to anaerobic incubation at 37 °C for 24 hours. SPME fibers were introduced by piercing the fibers through the septa insert of the lids and making sure that the fibers were exposed to volatile compounds present in the headspace without touching the media containing the bacteria. An internal standard was introduced before sampling using 1 μL of the standard solution (10 mL/L of decane-d22 in ethanol) per jar.

Anal sac samples. For the anal sac samples, single swabs (two containing anal sac fluid sample and one unused control swab) were placed individually into single septa screw cap jars that contained two SPME fibers. After a 24 hour incubation period, the SPMEs were removed. Then we performed a liquid extraction of volatiles by adding 20 mL of methanol to the jars and letting them sit for 24 hours.

GC-MS analysis

Chromatography occurred on a 7890 GC (Agilent Technologies Inc., Santa Clara, CA) with a ZB-WAX 30 m × 250 μm capillary column, coated with a 0.25 μm film stationary phase (Part 7HG-G007-11, 100% polyethylene glycol from Phenomenex, Torrance, CA) equivalent to DB-Wax or Carbowax. Helium was used as the carrier gas at 1 ml/min in constant flow mode. The inlet was set to 260 °C and SPMEs were splitlessly desorbed during the run. The oven temperature was programmed to increase from 40 °C (held for 5 minutes) to 110 °C at a rate of 5 °C per minute, and raise to 250 °C (held for 10 min) at a rate of 40 °C per minute. A transfer line set at 250 °C led to a 5977A mass spectrometer (Agilent Technologies Inc., Santa Clara, CA) with a solvent delay of 5 minutes. The MS swept from 50 to 500 m/z. The mass spectrometer was operated in the selected scan mode. The MS source was set to 230 °C and the MS quad set to 150 °C. A standard mix of C₈-C₂₀ alkanes was analyzed to calculate the Kovats Retention Indices and to monitor control of the instruments.

Methanolic extract of cat anal secretion was analyzed by GC-MS as tert-butyldimethylsilyl (TBDMS) derivative. 2 mL out of the 20 mL was used in the analysis. Samples were placed in glass conical vials and dried, reacted with a mixture of 50 μL N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA; Sigma-Aldrich Co. LLC., St Louis, MO, U.S.A.) and 50 μL acetonitrile at 60 °C for an hour. The derivatized anal sac solution was injected in duplicates into the GC-MS.

GC-MS data analysis workflow

MassHunter Profinder B.08.00 (Agilent Technologies Inc.) was used to deconvolute, integrate and align the data. Peaks with amplitudes of less than 1000 counts were ignored. Compounds must have been present in at least 60% of replicates from one treatment to be included in statistical analyses. Peak areas were normalized to the internal standard peak area of each data file. Furthermore, a VOC from a bacterial isolate or anal swab sample must have been, on average, three times greater than the respective controls (media blanks, etc.) to be included in this analysis. Tentative compound identification was based on the combined comparing mass spectra to the NIST 2014 Library and by a comparison of the calculated matching of standard alkane retention indices (LRI) values, when available.

Anal sac secretion constituents and bacteria isolates headspace SPME

We tentatively identified 127 compounds from the domestic cat anal gland secretion. Out of 127 tentatively identified compounds, 89 compounds were found in liquid extraction of anal secretion after TBDMS derivatization (S1 Table), and 52 compounds were measured by SPME-GC-MS in the anal sac secretion. These compounds were tentatively identified on the basis of the precise interpretation of its accurate mass spectra, MS fragmentation, and Kovats index information. These VOC metabolites were identified in the following compound chemical classes: heterocyclic compounds (12%), alcohols (16%), fatty acids (17%), ketones (11%), aromatic carbons (13%), amines (9%), aldehydes (7%), esters (6%).

A total of 67 unique compounds were tentatively identified from the SPME analysis of the three bacterial isolates, with some compounds being found in more than one isolate (19 compounds from B. fragilis UCD-AAL1, 44 compounds from Tessaracoccus sp. UCD-MLA, and 23 compounds from F. magna UCD-MLG) (Table 3). Among these 67 compounds, 52 compounds were also found in the anal sac secretion. 11 compounds (octan-1-ol, 1-(H)-indole, nonanoic acid, pentadecanoic acid, toluene, trans-2-pentenoic acid, non-2-enal, tetradecanal, n-hexadecanoic acid, octadecanoic acid, and (Z)-docos-13-enoic acid) found in the anal sac secretion have also been reported in other mammalian anal sac secretions [10,13,14,17,19,20,26,27]. Octan-1-ol and 1-(H)-indole were found in the anal sac secretion and in all three bacterial isolates. Octan-1-ol is a compound that produces a pungent odor and was previously reported in C. lupus anal sac secretion [26]. 1-(H)-indole is known to be widely distributed in the natural environment and can be produced by a variety of bacteria that have a strong fecal odor. 1-(H)-indole has also been found in various mammalian anal secretions, such as V. vulpes [61], F. catus [20], A. melanoleuca [13], C. lupus [26], and M. putorius furo [10]. Toluene is an aromatic compound found naturally in petroleum and coal, and is a major component of gasoline [62]. Raymer et. al. reported toluene found in C. lupus [26]. Although anaerobic bacterial toluene degradation is a well-known pathway [63], toluene biosynthesis is less common in bacteria. It is possible that our tentatively identified result could be a different compound but is likely to contain the alkyl benzene structure. Non-2-enal, and tetradecanal are chain aldehyde compounds. Non-2-enal was previously found in C. lupus [26] and tetradecanal was found in both S. suricatta [19,27], and A. melanoleuca [17] anal sac secretions. It is likely these aldehydes are formed by bacterial oxidation of a ubiquitous fatty acid such as oleic acid [64]. Out of 52 compounds, six fatty acids were tentatively identified from the anal sac secretion here; nonanoic acid, pentadecanoic acid, trans-2-pentenoic acid, n-hexadecanoic acid, octadecanoic acid, and (Z)-docos-13-enoic acid. Fatty acids are one of the most common compound groups known to be present in various animal anal sac secretions [13,14,16,17,19,27] and all of the fatty acids found in the anal sac secretion have been previously reported in other mammalian anal sac secretions. Nonanoic acid is a nine carbon fatty acid known to have unpleasant rancid odor, and previously found in S. suricatta anal sac secretions [19]. Pentadecanoic acid was found in P. leo [27], S. suricatta [19], and A. melanoleuca [13,17] anal sac secretions. Trans-2-pentenoic acid was found in H. hyaena [14] anal sac secretions. N-hexadecanoic acid was found in P. leo [27], S. suricatta [19], A. melanoleuca [17] anal sac secretions. (Z)-docos-13-enoic acid was found in A. melanoleuca [13,17] anal sac secretions. Fatty acids are a very common group biosynthesized by variety of organisms. The biogenesis typically starts with acetyl CoA, which is extended with malonate units to ultimately assemble fatty acids containing an even number of carbons [64]. All six fatty acids found in the anal sac secretion were also found in the Tessaracoccus sp. UCD-MLA culture. It is possible that the highly abundant (in the sample here) Tessaracoccus is largely contributing in the production of fatty acid compounds in this anal sac.

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Table 3. Tentatively identified compounds (compound name and formula listed) from cultures and anal sac.

The presence of the compound is indicated by a ‘+’ in the column corresponding to each sample: Bengal cat anal sac secretion chemical profile (“Anal sac”) and headspace solid phase microextraction (SPME) for cultures of B. fragilis UCD-AAL1 (“Bf”), Tessaracoccus sp. UCD-MLA (“Tess”), and F. magna UCD-MLG (“Fm”)).

https://doi.org/10.1371/journal.pone.0216846.t003

41 compounds found in the Bengal cat anal sac secretion have not been previously described in an anal sac secretion. Cyclohexanone and dimethyl trisulfide were found from the anal sac secretion and also from all three bacteria isolates. Cyclohexanone is known to be generated by the cyclohexanol degradation metabolomic pathway, which is widely used by bacteria [65]. Including dimethyl trisulfide, volatile sulfur compounds (hydrogen sulfide, methanethiol, dimethyl sulfide, dimethyl disulfide) such methionine derived volatiles are often generated by bacteria [64] and have also been reported to be produced by the Bacteroides family [66]. Hydrocarbons, aliphatic alcohols, and ketones are volatiles most likely formed by modification of products of the fatty acid biosynthetic pathway [64]. Alkanes and methyl ketones are often known to be produced from decarboxylation in bacteria [64]. Methyl ketone group compounds (2-methyl cyclopentanone, 1-(1,3-thiazol-2-yl)ethanone, 1-phenylethan-1-one) were tentatively identified from the cat anal sac secretion and at least one of the bacteria isolates. Another final modification process of the fatty acid metabolomic pathway is the reduction of carboxy groups to aldehydes and into aliphatic alcohols. Several aliphatic alcohols (decan-1-ol, butan-1-ol, 2-methylhexadecan-1-ol, 2-hexyldecan-1-ol) were tentatively identified from the anal sac secretion and from bacteria isolates. Butan-1-ol and its bishomologues up to C16 have been found in different combinations in several bacteria [64,67,68]. Aromatic compounds are common natural products in plants but also known to be produced by bacteria [64]. Especially among the aromatic alcohols, such phenols, 2-penylethanol is one of the most aromatic compounds produced by diverse bacteria. In this present study, aromatic alcohols, such as benzyl alcohol, and 3,5-ditert-butylphenol were tentatively identified from both anal sac and bacterial isolates. These aromatic alcohols can be assumed to be generated by the shikimate pathway which is present only in microorganisms and plants, never in animals [69].