Microbial community response to hydrocarbon exposure in iron oxide mats: an environmental study.

Hydrocarbon pollution is a widespread issue in both groundwater and surface-water systems; however, research on remediation at the interface of these two systems is limited. This interface is the oxic-anoxic boundary, where hydrocarbon pollutant from contaminated groundwaters flows into surface waters and iron mats are formed by microaerophilic iron-oxidizing bacteria. Iron mats are highly chemically adsorptive and host a diverse community of microbes. To elucidate the effect of hydrocarbon exposure on iron mat geochemistry and microbial community structure and function, we sampled iron mats both upstream and downstream from a leaking underground storage tank. Hydrocarbon-exposed iron mats had significantly higher concentrations of oxidized iron and significantly lower dissolved organic carbon and total dissolved phosphate than unexposed iron mats. A strong negative correlation between dissolved phosphate and benzene was observed in the hydrocarbon-exposed iron mats and water samples. There were positive correlations between iron and other hydrocarbons with benzene in the hydrocarbon-exposed iron mats, which was unique from water samples. The hydrocarbon-exposed iron mats represented two types, flocculent and seep, which had significantly different concentrations of iron, hydrocarbons, and phosphate, indicating that iron mat is also an important context in studies of freshwater mats. Using constrained ordination, we found the best predictors for community structure to be dissolved oxygen, pH, and benzene. Alpha diversity and evenness were significantly lower in hydrocarbon-exposed iron mats than unexposed mats. Using 16S rDNA amplicon sequences, we found evidence of three putative nitrate-reducing iron-oxidizing taxa in microaerophile-dominated iron mats (Azospira, Paracoccus, and Thermomonas). 16S rDNA amplicons also indicated the presence of taxa that are associated with hydrocarbon degradation. Benzene remediation-associated genes were found using metagenomic analysis both in exposed and unexposed iron mats. Furthermore, the results indicated that season (summer vs. spring) exacerbates the negative effect of hydrocarbon exposure on community diversity and evenness and led to the increased abundance of numerous OTUs. This study represents the first of its kind to attempt to understand how contaminant exposure, specifically hydrocarbons, influences the geochemistry and microbial community of freshwater iron mats and further develops our understanding of hydrocarbon remediation at the land-water interface.

Microbial communities are indicators of parasite infection status.

Growing evidence suggests that microbiomes have been shaping the evolutionary pathways of macroorganisms for millennia and that these tiny symbionts can influence, and possibly even control, species interactions like host-parasite relationships. Yet, while studies have investigated host-parasites and microbiomes separately, little has been done to understand all three groups synergistically. Here, we collected infected and uninfected Eurypanopeus depressus crab hosts from a coastal North Carolina oyster reef three times over 4 months. Infected crabs demonstrated an external stage of the rhizocephalan parasite, Loxothylacus panopaei. Community analyses revealed that microbial richness and diversity were significantly different among tissue types (uninfected crab, infected crab, parasite externae and parasite larvae) and over time (summer and fall). Specifically, the microbial communities from parasite externae and larvae had similar microbiomes that were consistent through time. Infected crabs demonstrated microbial communities spanning those of their host and parasite, while uninfected crabs showed more distinctive communities with greater variability over time. Microbial communities were also found to be indicators of early-stage infections. Resolving the microbial community composition of a host and its parasite is an important step in understanding the microbiome's role in the host-parasite relationship and determining how this tripartite relationship impacts coevolutionary processes.

Asymmetric Ocular Vestibular Evoked Myogenic Potentials in Pediatric Vestibular Migraine.

OBJECTIVE: Although ocular vestibular evoked myogenic potentials (oVEMP) abnormalities have been associated with vestibular migraine (VM) in adults, no studies have evaluated this in pediatric patients with VM. oVEMP asymmetry with normal cervical VEMP (cVEMP) findings may be a reliable VM biomarker in adults. We characterize VEMP results among pediatric patients with VM and benign recurrent vertigo of childhood (BRVC), a migraine precursor, and compare these results with VEMP findings from pediatric patients with nonmigrainous vestibular diagnoses. METHODS: Four hundred seventy-four pediatric patients were evaluated over a 3-year period in a multidisciplinary pediatric vestibular clinic, 139 of whom met the inclusion criteria. Records were reviewed for history, audiometry, and vestibular testing results. VEMP testing was performed with a 500-Hz tone burst. Based on adult normative data, oVEMP asymmetry was defined as greater than 33% interaural difference; cVEMP asymmetry was defined as greater than 41%. RESULTS: Eighty subjects had VM or BRVC (mean [standard deviation] 12.8 [3.8] yr; range, 4.3-18.2 yr). Fifty-nine subjects received vestibular diagnoses other than VM or BRVC (nonmigrainous group; mean [standard deviation] age, 13.0 [3.6] yr; range, 5.14-18.9 yr). A greater proportion of the VM/BRVC group demonstrated oVEMP asymmetry with normal cVEMP as compared with the nonmigrainous group (22.5% versus 10.1%; p < 0.05). Zero subjects in the VM/BRVC group demonstrated cVEMP asymmetry versus three subjects (4.9%) of the nonmigrainous group. CONCLUSIONS: VM and BRVC are notable causes of migraine-related vertigo among pediatric patients. Of VM/BRVC patients, 22.5% had oVEMP asymmetry with normal cVEMP. Similar to the adult VM population, this finding may be a useful biomarker in the right clinical setting for pediatric VM. LEVEL OF EVIDENCE: 4.

Coastal Microbial Communities Disrupted During the 2018 Hurricane Season in Outer Banks, North Carolina.

Hurricane frequencies and intensities are expected to increase under warming climate scenarios, increasing potential to disrupt microbial communities from steady-state conditions and alter ecosystem function. This study shows the impact of hurricane season on microbial community dynamics within the barrier island system of Outer Banks, North Carolina. We found that the passage of two sequential energetic hurricanes in 2018 (Florence and Michael) were correlated with shifts in total and active (DNA and RNA) portions of bacterial communities but not in archaeal communities, and within surface waters but not within the sediment. These microbial community shifts were distinct from non-hurricane season conditions, suggesting significant implications for nutrient cycling in nearshore and offshore environments. Hurricane-influenced marine sites in the coastal North Atlantic region had lower microbial community evenness and Shannon diversity, in addition to increased relative abundance of copiotrophic microbes compared to non-hurricane conditions. The abundance of functional genes associated with carbon and nitrogen cycling pathways were also correlated with the storm season, potentially shifting microbial communities at offshore sites from autotroph-dominated to heterotroph-dominated and leading to impacts on local carbon budgets. Understanding the geographic- and system-dependent responses of coastal microbial communities to extreme storm disturbances is critical for predicting impacts to nutrient cycling and ecosystem stability in current and future climate scenarios.

Orange leads to black: evaluating the efficacy of co-culturing iron-oxidizing and sulfate-reducing bacteria to discern ecological relationships.

Two global cycles, iron and sulfur, are critically interconnected in estuarine environments by microbiological actors. To this point, the methods of laboratory study of this interaction have been limited. Here we propose a methodology for co-culturing from numerous coastal environments, from the same source inocula, iron-oxidizing and sulfate-reducing bacteria. The use of same source inocula is largely beneficial to understand real-world interactions that are likely occurring in situ. Through the use of this methodology, the ecological interactions between these groups can be studied in a more controlled environment. Here, we characterize the oxygen and hydrogen sulfide concentrations using microelectrode depth profiling in the co-cultures of iron-oxidizing bacteria and sulfate-reducing bacteria. These results suggest that while oxygen drives the relationship between these organisms and sulfate-reducers are reliant on iron-oxidizers in this culture to create an anoxic environment, there is likely another environmental driver that also influences the interaction as the two remain spatially distinct, as trends in FeS precipitation changed within the anoxic zone relative to the presence of Fe(III) oxyhydroxides. Understanding the relationship between iron-oxidizing and sulfate-reducing bacteria will ultimately have implications for understanding microbial cycling in estuarine environments as well as in processes such as controlling microbially influenced corrosion.

Iron Flocs and the Three Domains: Microbial Interactions in Freshwater Iron Mats.

Freshwater iron mats are dynamic geochemical environments with broad ecological diversity, primarily formed by the iron-oxidizing bacteria. The community features functional groups involved in biogeochemical cycles for iron, sulfur, carbon, and nitrogen. Despite this complexity, iron mat communities provide an excellent model system for exploring microbial ecological interactions and ecological theories in situ Syntrophies and competition between the functional groups in iron mats, how they connect cycles, and the maintenance of these communities by taxons outside bacteria (the eukaryota, archaea, and viruses) have been largely unstudied. Here, we review what is currently known about freshwater iron mat communities, the taxa that reside there, and the interactions between these organisms, and we propose ways in which future studies may uncover exciting new discoveries. For example, the archaea in these mats may play a greater role than previously thought as they are diverse and widespread in iron mats based on 16S rRNA genes and include methanogenic taxa. Studies with a holistic view of the iron mat community members focusing on their diverse interactions will expand our understanding of community functions, such as those involved in pollution removal. To begin addressing questions regarding the fundamental interactions and to identify the conditions in which they occur, more laboratory culturing techniques and coculture studies, more network and keystone species analyses, and the expansion of studies to more freshwater iron mat systems are necessary. Increasingly accessible bioinformatic, geochemical, and culturing tools now open avenues to address the questions that we pose herein.

Introducing a "core steel microbiome" and community functional analysis associated with microbially influenced corrosion.

Microorganisms attached to aquatic steel structures play key roles in nutrient cycling and structural degradation processes. Corrosion-causing microbes are often the focus of studies involving microbially influenced corrosion, yet the roles of remaining community members remain unclear. This study characterizes the composition and functional potential of a 'core steel microbiome' across stainless steel types (304 and 316) and historic shipwreck steel along salinity gradients in North Carolina estuaries. We found higher phylogenetic evenness and diversity on steel surfaces compared to sediment, and at lower salinities. The core steel microbiome was composed of heterotrophic generalist taxa, and community composition was most strongly influenced by salinity. Substrate type was a secondary factor becoming more influential at higher salinities. The core steel microbiome included members of Sphingobacteriia, Cytophagia, Anaerolineaceae, Verrucomicrobiaceae, Chitinophagaceae, and Rheinheimera. While salinity differences led to phylogenetic separations across microbial community assemblages, functional genes were conserved across salinity and steel type. Generalist taxa on steel surfaces likely provide functional stability and biofilm protection for the community with limited functional trade-offs compared to surrounding environments. Further, characterization of a core steel microbiome increases the understanding of these complex steel surface microbial communities and their similarities to core microbiomes in other environments.

A Shallow Water Ferrous-Hulled Shipwreck Reveals a Distinct Microbial Community.

Shipwrecks act as artificial reefs and provide a solid surface in aquatic systems for many different forms of life to attach to, especially microbial communities, making them a hotspot of biogeochemical cycling. Depending on the microbial community and surrounding environment, they may either contribute to the wreck's preservation or deterioration. Even within a single wreck, preservation and deterioration processes may vary, suggesting that the microbial community may also vary. This study aimed to identify the differences through widespread sampling of the microbial communities associated with the Pappy Lane shipwreck (NC shipwreck site #PAS0001), a shallow water ferrous-hulled shipwreck in Pamlico Sound, North Carolina to determine if there are differences across the wreck as well as from its surrounding environment. Loose shipwreck debris, drilled shipcores, surrounding sediment, and seawater samples were collected from the Pappy Lane shipwreck to characterize the microbial communities on and around the shipwreck. Results indicated that the shipwreck samples were more similar to each other than the surrounding sediment and aquatic environments suggesting they have made a specialized niche associated with the shipwreck. There were differences between the microbial community across the shipwreck, including between visibly corroded and non-corroded shipwreck debris pieces. Relative abundance estimates for neutrophilic iron-oxidizing bacteria (FeOB), an organism that may contribute to deterioration through biocorrosion, revealed they are present across the shipwreck and at highest abundance on the samples containing visible corrosion products. Zetaproteobacteria, a known class of marine iron-oxidizers, were also found in higher abundance on shipwreck samples with visible corrosion. A novel Zetaproteobacteria strain, Mariprofundus ferrooxydans O1, was isolated from one of the shipwreck pieces and its genome analyzed to elucidate the functional potential of the organism. In addition to iron oxidation pathways, the isolate has the genomic potential to perform carbon fixation in both high and low oxygen environments, as well as perform nitrogen fixation, contributing to the overall biogeochemical cycling of nutrients and metals in the shipwreck ecosystem. By understanding the microbial communities associated with shallow water ferrous-hulled shipwrecks, better management strategies and preservation plans can be put into place to preserve these artificial reefs and non-renewable cultural resources.

The effects of temperature on Bosmina longirostris susceptibility to microcystin-LR acute toxicity.

Harmful algal blooms are an ongoing threat to many aquatic systems throughout the world. In the Chowan River, North Carolina, the frequency of toxin producing Microcystis aeruginosa blooms has increased since 1975 along with an average 0.71°C rise in water temperature. The combined effect of microcystin-LR toxin and rising temperatures on a dominant zooplankter in the system, Bosmina longirostris, was the focus of this study. Laboratory studies were conducted to determine how microcystin-LR, produced from M. aeruginosa blooms, affected B. longirostris mortality under different temperature regimes. At 25°C, the LC50 for B. longirostris was 26.3 µg L-1 suggesting that B. longirostris can survive typical current bloom microcystin-LR concentrations ranging 0.1µg L-1 to 2.0 µg L-1, but would be susceptible to higher concentrations they may be periodically exposed to. Mortality was assessed at a constant microcystin-LR concentration of 26.3 µg L-1 over 15-35°C, and it was found that B. longirostris mortality increased at higher temperatures. B. longirostris mortality increased approximately 18% due to microcystin-LR alone over 2°C between 25°C and 27°C when exposed to the LC50 concentration. The increased prevalence of toxic M. aeruginosa blooms and increasing temperatures due to climate change may reduce B. longirostris populations, potentially affecting larval fish and fisheries in the Chowan River, North Carolina.

Environmental Evidence for and Genomic Insight into the Preference of Iron-Oxidizing Bacteria for More-Corrosion-Resistant Stainless Steel at Higher Salinities.

Iron-oxidizing bacteria (FeOB) are some of the initial colonizing organisms during microbially influenced corrosion of steel infrastructure. To better understand the abiotic conditions under which FeOB colonize steel, an environmental study was conducted to determine the effects of salinity, temperature, dissolved oxygen levels, and steel type on FeOB colonization. Stainless steel (304 and 316 [i.e., 304SS and 316SS]) was used to determine the potential susceptibility of these specialized corrosion-resistant steels. Steel coupon deployments along salinity gradients in two river systems revealed attachment by FeOB at all sites, with greater abundance of FeOB at higher salinities and on 316SS, compared to 304SS. This may be due to the presence of molybdenum in 316SS, potentially providing a selective advantage for FeOB colonization. A novel Zetaproteobacteria species, Mariprofundus erugo, was isolated from these stainless steel samples. Genes for molybdenum utilization and uptake and reactive oxygen species protection were found within its genome, supporting the evidence from our FeOB abundance data; they may represent adaptations of FeOB for colonization of surfaces of anthropogenic iron sources such as stainless steel. These results reveal environmental conditions under which FeOB colonize steel surfaces most abundantly, and they provide the framework needed to develop biocorrosion prevention strategies for stainless steel infrastructure in coastal estuarine areas.IMPORTANCE Colonization of FeOB on corrosion-resistant stainless steel types (304SS and 316SS) has been quantified from environmental deployments along salinity gradients in estuarine environments. Greater FeOB abundance at higher salinities and on the more-corrosion-resistant 316SS suggests that there may be a higher risk of biocorrosion at higher salinities and there may be a selective advantage from certain stainless steel alloy metals, such as molybdenum, for FeOB colonization. A novel species of FeOB described here was isolated from our stainless steel coupon deployments, and its genome sequence supports our environmental data, as genes involved in the potential selectiveness toward surface colonization of stainless steel might lead to higher rates of biocorrosion of manmade aquatic infrastructure. These combined results provide environmental constraints for FeOB colonization on anthropogenic iron sources and build on previous frameworks for biocorrosion prevention strategies.

Core versus specialty rotations do not affect students' surgical development.

BACKGROUND: Research is scarce on how the diversity of surgical rotations affects students. We sought to assess the effect of core rotations compared to specialty rotations on students' development. METHODS: Students were given a suturing workshop at the beginning of their surgical clerkship along with a questionnaire. They performed both a simple and a complex suturing task at the beginning and end of the 2-month clerkship. The students were divided into 2 groups based on their surgical rotations. Technical skill and exam scores were compared. RESULTS: Thirty-eight students were included in the study. Objective scores increased for the simple task (14.2, standard deviation 4.5 vs 16.4, standard deviation 4.2, P = .04) and the complex task (12.9, standard deviation 5.3 vs 16.5, standard deviation 4.1, P < .01). Times decreased for the simple task (5.1, standard deviation 1.8 vs 4.1, standard deviation 1.3, min, P < .01) and the complex task (7.9, standard deviation 2.7 vs 6.3, standard deviation 1.5, min, P < .01). Using multivariate analysis, we found that reported hours in the operating room per week and previous hands-on experience affected proficiency of the simple suturing task only. Sixteen students had predominantly core surgical rotations. When compared to the 22 students with more specialty rotations, the only difference was gender (87.5% male vs 50.0% male, P = 0.02). There was no significant difference in the completion times (P = .96, .82), the objective scores (P = .06, .120), the written exam scores (P = .57), or the oral exam scores (P = .89). CONCLUSION: In this small study, it was found that the type of students' rotations does not affect surgical skill or knowledge acquisition.

Carbon-dependent chromate toxicity mechanism in an environmental Arthrobacter isolate.

Arthrobacter spp. are widespread in soil systems and well-known for their Cr(VI) reduction capabilities making them attractive candidates for in situ bioremediation efforts. Cellulose drives carbon flow in soil systems; yet, most laboratory studies evaluate Arthrobacter-Cr(VI) interactions solely with nutrient-rich media or glucose. This study aims to determine how various cellulose degradation products and biostimulation substrates influence Cr(VI) toxicity, reduction, and microbial growth of an environmental Arthrobacter sp. isolate. Laboratory culture-based studies suggest there is a carbon-dependent Cr(VI) toxicity mechanism that affects subsequent Cr(VI) reduction by strain LLW01. Strain LLW01 could only grow in the presence of, and reduce, 50 µM Cr(VI) when glucose or lactate were provided. Compared to lactate, Cr(VI) was at least 30-fold and 10-fold more toxic when ethanol or butyrate was the sole carbon source, respectively. The addition of sulfate mitigated toxicity somewhat, but had no effect on the extent of Cr(VI) reduction. Cell viability studies indicated that a small fraction of cells were viable after 8 days suggesting cell growth and subsequent Cr(VI) reduction may resume. These results suggest when designing bioremediation strategies with Arthrobacter spp. such as strain LLW01, carbon sources such as glucose and lactate should be considered over ethanol and butyrate.

Novel Pelagic Iron-Oxidizing Zetaproteobacteria from the Chesapeake Bay Oxic-Anoxic Transition Zone.

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O2] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O2, as indicated by the presence of genes encoding aa3-type cytochrome c oxidases, but not the cbb3-type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II).

Genomic exploration of individual giant ocean viruses.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

A pearl in the ear: Intracranial complications of pediatric cholesteatomas.

A nine-year-old male had a cholesteatoma of the mastoid and middle ear found incidentally after myringotomy tube placement. Associated asymptomatic sigmoid plate dehiscence with sinus invasion or thrombosis and ossicular chain destruction complicated his case. He had canal wall down tympanomastoidectomy and was followed for 4.5 years. Disease recurrence necessitated revision. Our case highlights an unusual clinical presentation, possible complications, and the aggressive quality of a benign lesion common in the pediatric population. To our knowledge, this is the first report of an asymptomatic lateral sinus obstruction secondary to an invasive cholesteatoma in this population.

Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs.

Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota.

Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population.

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

Management of First Branchial Cleft Anomalies via a Cartilage-Splitting Technique.

First branchial cleft anomalies are uncommon lesions that often present as periauricular infections. They have high recurrence rates, due in part to scarring secondary to prior infections and their management. These lesions have a close relationship with the facial nerve, and most authors recommend its identification and dissection because of this relationship. Nonetheless, facial nerve palsy has been reported in up to 15% of cases. We describe a novel technique for the management of first branchial cleft anomalies. Such lesions that presented in an infra- or postauricular location were approached via an incision through the cartilage of the pinna, between the tragus and antitragus. This technique affords direct access to the lesion without the need for facial nerve dissection. Six patients were treated. Five had prior surgery, including 3 with previous attempts at excision. There were no complications. The median follow-up was 35 months. One patient developed a recurrence.

The role of gastric pepsin in the inflammatory cascade of pediatric otitis media.

IMPORTANCE: Otitis media is characterized as an ongoing inflammation with accumulation of an effusion in the middle ear cleft. The molecular mechanisms underlying the pathogenesis, particularly the inflammatory response, remain largely unknown. We hypothesize that aspiration of gastric contents into the nasopharynx may be responsible for the initiation of the inflammatory process or aggravate a preexisting condition. OBJECTIVE: To investigate the correlation of gastric pepsin A with inflammatory cytokines, bacterial infection, and clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: Prospective study of 129 pediatric patients undergoing myringotomy with tube placement for otitis media at a tertiary care pediatric hospital. MAIN OUTCOMES AND MEASURES: Ear samples were tested for pepsin A; cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor; and bacterial culture inoculation. Data were analyzed by descriptive statistics and regression analysis to identify risk factors for the presence of pepsin A and to correlate pepsin A levels with cytokine levels, infection status, and clinical outcomes. RESULTS: Of the 129 patients, 199 ear samples were obtained; 82 samples (41%) and 64 patients (50%) were positive for pepsin A as measured by immunoassay. Pepsin A positivity correlated with age younger than 3.0 years (mean [SD], 2.3 [2.1] years in the positive group vs 3.3 [3.0] years in the negative group) and with all 3 cytokine levels (mean [SD] tumor necrosis factor, 29.5 [45.9] pg/mL in the positive group vs 13.2 [21.6] pg/mL in the negative group; IL-6, 6791.7 [9389.1] pg/mL in the positive group vs 2849.9 [4066.3] pg/mL in the negative group; and IL-8, 6828.2 [8122.3] pg/mL in the positive group vs 2925.1 [3364.5] pg/mL in the negative group [all P < .05]); however, logistic regression analysis showed that only IL-8 (odds ratio, 3.96; 95% CI, 1.3-12.0; P = .02) and age (odds ratio, 3.83; 95% CI, 1.2-12.7; P = .03) were significant independent variables. No statistically significant association was found with other parameters. Multiple linear regressions revealed that the levels of pepsin A were correlated with IL-8 levels (R2 = 0.248; P < .001) and the need for second or third tubes 6 to 12 months after the first (R2 = 0.102; P = .006). The presence of pepsin A in the middle ear was not associated with increased bacterial infection. Interleukin 8 was independent and significantly associated with both pepsin A levels and bacterial infection (R2 = 0.144 and 0.263, respectively; P = .001 for both). CONCLUSIONS AND RELEVANCE: Extraesophageal reflux as indicated by the presence of pepsin A is closely involved in the middle ear inflammatory process and may worsen the disease in some children; however, a proof of cause and effect between extraesophageal reflux and middle ear inflammation requires further investigation.

Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount.

The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.