Volcanic caves are filled with colorful microbial mats on the walls and ceilings. These volcanic caves are found worldwide, and studies are finding vast bacteria diversity within these caves. One group of bacteria that can be abundant in volcanic caves, as well as other caves, is Actinobacteria. As Actinobacteria are valued for their ability to produce a variety of secondary metabolites, rare and novel Actinobacteria are being sought in underexplored environments. The abundance of novel Actinobacteria in volcanic caves makes this environment an excellent location to study these bacteria. Scanning electron microscopy (SEM) from several volcanic caves worldwide revealed diversity in the morphologies present. Spores, coccoid and filamentous cells, many with hair-like or knobby extensions, were some of the microbial structures observed within the microbial mat samples. In addition, the SEM study pointed out that these features figure prominently in both constructive and destructive mineral processes. To further investigate this diversity, we conducted both Sanger sequencing and 454 pyrosequencing of the Actinobacteria in volcanic caves from four locations, two islands in the Azores, Portugal and Hawai`i and New Mexico, USA. This comparison represents one of the largest sequencing efforts of Actinobacteria in volcanic caves to date. The diversity was shown to be dominated by Actinomycetales, but also included several newly described orders, such as Euzebyales, and Gaiellales. Sixty-two percent of the clones from the four locations shared less than 97% similarity to known sequences, and nearly 71% of the clones were singletons, supporting the commonly held belief that volcanic caves are an untapped resource for novel and rare Actinobacteria. The amplicon libraries depicted a wider view of the microbial diversity in Azorean volcanic caves revealing three additional orders, Rubrobacterales, Solirubrobacterales and Coriobacteriales. Studies of microbial ecology in volcanic caves are still very limited. To rectify this deficiency, the results from our study help fill in the gaps in our knowledge of actinobacterial diversity and their potential roles in the volcanic cave ecosystems.

The terrestrial subsurface microbiome has gained considerable amount of interests in the recent years because of its rich potential resource for biomining novel genes coding for metabolites possessing antimicrobial activities. In our previous study, we identified two Streptomyces isolates, designated as ICC1 and ICC4, from the Iron Curtain Cave, Chilliwack, Canada that exhibited antagonistic activities against the multidrug resistant strains of Escherichia coli. In this study, the genomes of these two isolates were sequenced by Illumina MiSeq, assembled and annotated. The genes associated with secondary metabolite production were identified and annotated using the bioinformatics platforms antiSMASH and BAGEL. ICC1 and ICC4 were then cultivated and ICC1 metabolome characterized by UHPLC-ESI-HRMS. The Global Natural Products Social Molecular Networking was used to identify metabolites based on the MS/MS spectral data. ICC1 and ICC4 showed a high level of sequence identity with the terrestrial bacteria Streptomyces lavendulae; however, they possess a greater secondary metabolite potential as estimated by the total number of identified biosynthetic gene clusters (BGCs). In particular, ICC1 and ICC4 had a greater number of polyketide and non-ribosomal peptide BGCs. The most frequently detected BGCs were those predicted to generate terpenes, small and low complexity dipeptides and lipids. Spectral analysis clearly identified a number of diketopiperazine products through matched reference spectra for cyclo (Leu-Pro), cyclo (Pro-Val) and cyclo [(4-hydroxyPro)-Leu]. One of the terpenes gene clusters predicted by antiSMASH possesses a seven-gene pathway consistent with diazepinomicin biosynthesis. This molecule contains a very rare core structure and its BGC, to date, has only been identified from a single bacterial genome. The tetrapeptide siderophore coelichelin BGC was unambiguously identified in the genome, however, the metabolite could not be identified from the culture extracts. Two type III polyketides, 2′, 5′ – dimethoxyflavone and nordentatin, were identified from the UHPLC-HRMS data of the aqueous and n-butanolic fractions of Streptomyces sp. ICC1, respectively. A BGC likely encoding these metabolites was predicted in both genomes. The predicted similarities in molecule production and genome shared by these two strains could be an indicative of a cooperative mode of living in extreme habitats instead of a competitive one. This secondary metabolite potential may contribute to the fitness of ICC1 and ICC4 in the Iron Curtain Cave.

Caves are extreme, often oligotrophic, environments that house diverse groups of microorganisms. Many of these microbes can perform microbiologically induced carbonate precipitation (MICP) to form crystalline secondary cave deposits known as speleothems. The urease family is a group of enzymes involved in MICP that catalyze the breakdown of urea, which is a source of energy, into ammonia and carbonate. Carbonate anions are effluxed to the extracellular surface of the bacterium where it then binds to environmental calcium to form calcium carbonate which then continues to grow in crystal form. Here, we studied bacterial communities from speleothems collected from the Iron Curtain Cave (ICC) in Chilliwack, B.C., Canada, to characterize these organisms and determine whether urease-positive (U+) bacteria were present in the cave and their potential impact on speleothem formation. The ICC is a carbonate cave located on the northside of Chipmunk Ridge, presenting a unique environment with high iron content sediment and limestone structures throughout. With six pools of water throughout the cave, the environment is highly humid, with temperatures ranging between 4 and 12°C depending on the time of year. Ninety-nine bacterial strains were isolated from popcorn (PCS) and soda straw (SSS) speleothems. These isolates were screened for urease enzymatic activity, with 11 candidates found to be urease-positive. After incubation, species-specific crystal morphologies were observed. Popcorn speleothem provided more bacterial diversity overall when compared to soda straw speleothem when examined under a culture-based method. Nearly twice as many U+ isolates were isolated from popcorn speleothems compared to soda straw speleothems. The U+ candidates were identified to the genus level by 16S rRNA analysis, and two isolates underwent whole-genome sequencing. Two novel species were identified as Sphingobacterium sp. PCS056 and Pseudarthrobacter sp. SSS035. Both isolates demonstrated the most crystal production as well as the most morphologically dissimilar crystal shapes in broth culture and were found to produce crystals as previously observed in both agar and broth media. The results from this study are consistent with the involvement of urease-positive bacteria isolated from the ICC in the formation of cave speleothems. 16S rRNA sequencing revealed a diverse set of microbes inhabiting the speleothems that have urease activity. Whole-genome sequencing of the two chosen isolates confirmed the presence of urease pathways, while revealing differences in urease pathway structure and number. This research contributes to understanding microbial-associated cave formation and degradation, with applications to cave conservation, microbiota composition, and their role in shaping the cave environment.

BACKGROUND: Moraxella catarrhalis is a commensal organism of the respiratory tract that has emerged as an important pathogen for a variety of upper and lower respiratory tract infections including otitis media and acute exacerbations of chronic bronchitis. Susceptibility testing of M catarrhalis is not routinely performed in most diagnostic laboratories; rather, a comment predicting susceptibility based on the literature is attached to the report. The most recent Canadian report on M catarrhalis antimicrobial susceptibility was published in 2003; therefore, a new study at this time was of interest and importance.OBJECTIVE: To determine the susceptibility of M catarrhalis isolates from British Columbia to amoxicillin‐clavulanate, doxycycline, clarithromycin, cefuroxime, levofloxacin and trimethoprimsulfamethoxazole.METHODS: A total of 117 clinical M catarrhalis isolates were isolated and tested from five Interior hospitals and two private laboratory centres in British Columbia between January and December 2012. Antibiotic susceptibility of M catarrhalis isolates was characterized using the Etest (E‐strip; bioMérieux, USA) according to Clinical Laboratory Standards Institute guidelines.RESULTS: All isolates were sensitive to amoxicillin‐clavulanate, doxycycline, clarithromycin, levofloxacin and trimethoprimsulfamethoxazole. One isolate was intermediately resistant to cefuroxime, representing a 99.15% sensitivity rate to the cephem agent. Cefuroxime minimum inhibitory concentrations (MICs) inhibiting 50% and 90% of organisms (MIC50 and MIC90) were highest among the antibiotics tested, and the MIC90 (3 μg/mL) of cefuroxime reached the Clinical Laboratory Standards Institute breakpoint of susceptibility.DISCUSSION: The antibiotic susceptibility of M catarrhalis isolates evaluated in the present study largely confirms the findings of previous surveillance studies performed in Canada. Cefuroxime MICs are in the high end of the sensitive range and the MIC50 and MIC90 observed in the present study are the highest ever reported in Canada.CONCLUSION: Although cefuroxime MICs in M catarrhalis are high, all agents tested showed antimicrobial activity, supporting their continued therapeutic and empirical use.