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Abstract Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. It is commonly assumed that biofilm cells are glued together by the matrix; however, how the specific biochemistry of matrix components affects the cell-matrix interactions and how these interactions vary during biofilm growth remain unclear. Here, we investigate cell-matrix interactions in Vibrio cholerae, the causative agent of cholera. We combine genetics, microscopy, simulations, and biochemical analyses to show that V. cholerae cells are not attracted to the main matrix component (Vibrio polysaccharide, VPS), but can be attached to each other and to the VPS network through surface-associated VPS and crosslinks formed by the protein Bap1. Downregulation of VPS production and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results shed light on the dynamics of diverse cell-matrix interactions as drivers of biofilm development.

ABSTRACT Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili (TFP)-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of TFP motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. Reduced invasion leads to the formation of denser and thicker S. aureus colonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies expand our understanding of how P. aeruginosa TFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.IMPORTANCEThe polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation of Pseudomonas aeruginosa and Staphylococcus aureus from airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils that P. aeruginosa is attracted to S. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition of P. aeruginosa motility and thus invasion, S. aureus colony architecture changes dramatically, whereby S. aureus is protected from P. aeruginosa antagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition.

ABSTRACTPredators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains of Escherichia coli and Salmonella enterica by competing a generalist T5-like phage able to infect both prey against P22vir, an S. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that the in vitro cost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest.IMPORTANCEThere is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and an in vitro system consisting of Escherichia coli, Salmonella enterica, a T5-like generalist phage, and the specialist phage P22vir to highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections.

IntroductionCoordinated alignment of the eyes during gaze fixation and eye movements are an important component of normal visual function. We have previously described the coordinated behavior of convergence eye movements and pupillary responses using a 0.1 Hz binocular disparity-driven sine profile and a step profile. The goal of this publication is to further characterize ocular vergence-pupil size coordination over a wider range of frequencies of ocular disparity stimulation in normal subjects.MethodsBinocular disparity stimulation is generated by presentation of independent targets to each eye on a virtual reality display, while eye movements and pupil size are measured by an embedded video-oculography system. This design allows us to study two complimentary analyses of this motion relationship. First, a macroscale analysis describes the vergence angle of the eyes in response to binocular disparity target movement and pupil area as a function of the observed vergence response. Second, a microscale analysis performs a piecewise linear decomposition of the vergence angle and pupil relationship to permit more nuanced findings.ResultsThese analyses identified three main features of controlled coupling of pupil and convergence eye movements. First, a near response relationship operates with increasing prevalence during convergence (relative to the “baseline” angle); the coupling is higher with increased convergence in this range. Second, the prevalence of “near response”-type coupling decreases monotonically in the diverging direction; the decrease persists after the targets move (converge back) from maximum divergence toward the baseline positions, with a minimum prevalence of near response segments near the baseline target position. Third, an opposite polarity pupil response is infrequent, but tends to be more prevalent when the vergence angles are at maximum convergence or divergence for a sinusoidal binocular disparity task.DiscussionWe suggest that the latter response is an exploratory “range-validation” when binocular disparity is relatively constant. In a broader sense, these findings describe operating characteristics of the near response in normal subjects and form a basis for quantitative assessments of function in conditions such as convergence insufficiency and mild traumatic brain injury.

BackgroundThird window syndrome is a vestibular-cochlear disorder in humans in which a third mobile window of the otic capsule creates changes to the flow of sound pressure energy through the perilymph/endolymph. The nature and location of this third mobile window can occur at many different sites (or multiple sites); however, the most common third mobile window is superior semicircular canal dehiscence (SSCD). There are two essential objective diagnostic characteristics needed to validate a model of SSCD: the creation of a pseudoconductive hearing loss and cVEMP increased amplitude and decreased threshold.MethodsAdult Mongolian gerbils (n = 36) received surgical fenestration of the superior semicircular canal of the left inner ear. ABR and c+VEMP testing were carried out prior to surgery and over acute (small 1 mm SSCD, 1–10 days) or prolonged (large 2 mm SSCD, 28 days) recovery. Because recovery of function occurred quickly, condenser brightfield stereomicroscopic examination of the dehiscence site was carried out for the small SSCD animals post-hoc and compared to both ABRs and c+VEMPs. Micro-CT analysis was also completed with representative samples of control, day 3 and 10 post-SSCD animals.ResultsThe SSCD created a significant worsening of hearing thresholds of the left ear; especially in the lower frequency domain (1–4 kHz). Left (EXP)/right (CTL) ear comparisons via ABR show significant worsening thresholds at the same frequency representations, which is a proxy for the human pseudoconductive hearing loss seen in SSCD. For the c+VEMP measurements, increased amplitude of the sound-induced response (N1 2.5 ms and P1 3.2 ms) was observed in animals that received larger fenestrations. As the bone regrew, the c+VEMP and ABR responses returned toward preoperative values. For small SSCD animals, micro-CT data show that progressive osteoneogenesis results in resurfacing of the SSCD without bony obliteration.ConclusionThe large (2 mm) SSCD used in our gerbil model results in similar electrophysiologic findings observed in patients with SSCD. The changes observed also reverse and return to baseline as the SSCD heals by bone resurfacing (with the lumen intact). Hence, this model does not require a second surgical procedure to plug the SSCD.

Abstract Objective Eye tracking technology has been employed in assessing ocular motor and vestibular function following vestibular and neurologic conditions, including traumatic brain injury (TBI). Assessments include tests that provide visual and motion (rotation) stimuli while recording horizontal, vertical, and torsional eye movements. While some of these tests have shown diagnostic promise in previous studies, their use in clinical practice is limited by the lack of normative data. The goal of this study was to construct normative reference ranges to be used when comparing patients' results. Methods Optokinetic response, subjective visual horizontal and vertical, and rotation tests were administered to male and female volunteers, ages 18‐45, who were free from neurological, vestibular disorders, or other head injuries. Tests were administered using either a rotatory chair or a portable virtual reality‐like goggle equipped with video‐oculography. Results Reference values for eye movements in response to different patterns of stimuli were analyzed from 290 to 449 participants. Analysis of gender (self‐reported) or age when grouped as pediatric (late adolescent; 18‐21 years of age) and adult (21‐45 years of age) revealed no effects on the test metrics. Data were pooled and presented for each test metric as the 95% reference interval (RI) with 90% confidence intervals (CI) on upper and lower limits of the RI. Conclusions This normative database can serve as a tool to aid in diagnosis, treatment, and/or rehabilitation protocols for vestibular and neurological conditions, including mild TBI (mTBI). This database has been cleared by the FDA for use in clinical practice (K192186). Level of evidence 2b

ABSTRACT The human pathogen Vibrio cholerae grows as biofilms, communities of cells encased in an extracellular matrix. When growing in biofilms, cells compete for resources and space. One common competitive mechanism among Gram-negative bacteria is the type six secretion system (T6SS), which can deliver toxic effector proteins into a diverse group of target cells, including other bacteria, phagocytic amoebas, and human macrophages. The response regulator VxrB positively regulates both biofilm matrix and T6SS gene expression. Here, we directly observe T6SS activity within biofilms, which results in improved competition with strains lacking the T6SS. VxrB significantly contributes to both attack and defense via T6SS, while also influencing competition via regulation of biofilm matrix production. We further determined that both Vibrio polysaccharide (VPS) and the biofilm matrix protein RbmA can protect cells from T6SS attack within mature biofilms. By varying the spatial mixing of predator and prey cells in biofilms, we show that a high degree of mixing favors T6SS predator strains and that the presence of extracellular DNA in V. cholerae biofilms is a signature of T6SS killing. VxrB therefore regulates both T6SS attack and matrix-based T6SS defense, to control antagonistic interactions and competition outcomes during mixed-strain biofilm formation. IMPORTANCE This work demonstrates that the Vibrio cholerae type six secretion system (T6SS) can actively kill prey strains within the interior of biofilm populations with substantial impact on population dynamics. We additionally show that the response regulator VxrB contributes to both T6SS killing and protection from T6SS killing within biofilms. Components of the biofilm matrix and the degree of spatial mixing among strains also strongly influence T6SS competition dynamics. T6SS killing within biofilms results in increased localized release of extracellular DNA, which serves as an additional matrix component. These findings collectively demonstrate that T6SS killing can contribute to competition within biofilms and that this competition depends on key regulators, matrix components, and the extent of spatial population mixture during biofilm growth.

ABSTRACT Alanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment, Aspergillus fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA, and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment, which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms, and chemical inhibition of alaA by the alanine aminotransferase inhibitor β-chloro-l-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describe a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs. IMPORTANCE Aspergillus fumigatus is a ubiquitous filamentous fungus that causes an array of diseases depending on the immune status of an individual, collectively termed aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) is limited and too often ineffective. This is in part due to A. fumigatus biofilm formation within the infection environment and the resulting emergent properties, particularly increased antifungal resistance. Thus, insights into biofilm formation and mechanisms driving increased antifungal drug resistance are critical for improving existing therapeutic strategies and development of novel antifungals. In this work, we describe an unexpected observation where alanine metabolism, via the alanine aminotransferase AlaA, is required for several aspects of A. fumigatus biofilm physiology, including resistance of A. fumigatus biofilms to the echinocandin class of antifungal drugs. Importantly, we observed that chemical inhibition of alanine aminotransferases is sufficient to increase echinocandin susceptibility and that loss of alaA increases susceptibility to echinocandin treatment in a murine model of IPA. AlaA is the first gene discovered in A. fumigatus that confers resistance to an antifungal drug specifically in a biofilm context.

In the wild, bacteria grow into structures called biofilms. Here the authors demonstrate that their spatial organization and heterogeneity depends on the interplay between fluid flow and single cell motility; this highlights the role of hydrodynamics in biofilm formation.

A lack of effective methods for sampling lizards in terrain that is inaccessible to human observers limits our knowledge of their ecology and conservation needs. Drones are increasingly being used in wildlife monitoring, but their potential use for surveying lizards has not been evaluated. We investigated: (1) the detectability of model lizards using a drone relative to a human observer, and (2) the response of four lizard species to an approaching drone in three habitat types. Model lizards placed in potential basking positions within a defined search area were detected by both the drone operator and human observer, but the probability of detection was lower with the drone. Jewelled geckos (Naultinus gemmeus) in shrubland and grand skinks (Oligosoma grande) in rocky habitats showed surprisingly little reaction to the approaching drone, enabling close approaches (means of 59 cm and 107 cm, respectively) and accurate species identification with photos taken by the drone camera. For highly patterned jewelled geckos, identification was also possible to individual level. However, the drone was unsuccessful at detecting two alpine skink species in a near-vertical cliff habitat. Collectively, our results suggest that drones have potential as a tool for detecting small-bodied lizards in habitats inaccessible to human observers.

There is an abundance of work on both P. aeruginosaC. albicans

The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated.

In late 2016, diplomats in Havana, Cuba, began presenting with a unique symptom complex after perceiving a strange noise and/or feeling a pressure field in their domicile. This report is a retrospective, quantitative analysis of video-oculography data of pupillary light reflex performance and binocular disparity-driven eye and pupil movements during the acute time period after the reported exposure. The patterns of response in these 19 individuals are markedly different than those seen in a group of individuals with the usual acute mild traumatic brain injury (17 subjects) and from 62 control subjects (21–60 years old) with no injury. Non-linear least squares regression was used to estimate the model parameters from the eye movement and the pupil measurements (1). Linear discriminant analysis was then used to identify a classifier for an objective discrimination of the groups with >91% accuracy and no confusion between the acute neurosensory findings among the members of the Havana diplomatic community and the subjects with acute mild traumatic brain injury. This pattern difference in eye and pupil behavior may be a useful screen to help objectively distinguish blunt trauma from Havana-type effects in the future and to guide the affected individuals to appropriate care.

ABSTRACT Encounters among bacteria and their viral predators (bacteriophages) are among the most common ecological interactions on Earth. These encounters are likely to occur with regularity inside surface-bound communities that microbes most often occupy in natural environments. Such communities, termed biofilms, are spatially constrained: interactions become limited to near neighbors, diffusion of solutes and particulates can be reduced, and there is pronounced heterogeneity in nutrient access and physiological state. It is appreciated from prior theoretical work that phage-bacteria interactions are fundamentally different in spatially structured contexts, as opposed to well-mixed liquid culture. Spatially structured communities are predicted to promote the protection of susceptible host cells from phage exposure, and thus weaken selection for phage resistance. The details and generality of this prediction in realistic biofilm environments, however, are not known. Here, we explore phage-host interactions using experiments and simulations that are tuned to represent the essential elements of biofilm communities. Our simulations show that in biofilms, phage-resistant cells—as their relative abundance increases—can protect clusters of susceptible cells from phage exposure, promoting the coexistence of susceptible and phage-resistant bacteria under a large array of conditions. We characterize the population dynamics underlying this coexistence, and we show that coexistence is recapitulated in an experimental model of biofilm growth measured with confocal microscopy. Our results provide a clear view into the dynamics of phage resistance in biofilms with single-cell resolution of the underlying cell-virion interactions, linking the predictions of canonical theory to realistic models and in vitro experiments of biofilm growth. IMPORTANCE In the natural environment, bacteria most often live in communities bound to one another by secreted adhesives. These communities, or biofilms, play a central role in biogeochemical cycling, microbiome functioning, wastewater treatment, and disease. Wherever there are bacteria, there are also viruses that attack them, called phages. Interactions between bacteria and phages are likely to occur ubiquitously in biofilms. We show here, using simulations and experiments, that biofilms will in most conditions allow phage-susceptible bacteria to be protected from phage exposure, if they are growing alongside other cells that are phage resistant. This result has implications for the fundamental ecology of phage-bacteria interactions, as well as the development of phage-based antimicrobial therapeutics.

Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae, where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae, the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae, which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.

Objective: This communication is the first assessment of outcomes after surgical repair of cochlea-facial nerve dehiscence (CFD) in a series of patients. Pre- and post-operative quantitative measurement of validated survey instruments, symptoms, diagnostic findings and anonymous video descriptions of symptoms in a cohort of 16 patients with CFD and third window syndrome (TWS) symptoms were systematically studied.Study design: Observational analytic case-control study.Setting: Quaternary referral center.Patients: Group 1 had 8 patients (5 children and 3 adults) with CFD and TWS who underwent surgical management using a previously described round window reinforcement technique. Group 2 had 8 patients (2 children and 6 adults) with CFD who did not have surgical intervention.Interventions: The Dizziness Handicap Inventory (DHI) and Headache Impact Test (HIT-6) were administered pre-operatively and post-operatively. In addition, diagnostic findings of comprehensive audiometry, cervical vestibular evoked myogenic potential (cVEMP) thresholds and electrocochleography (ECoG) were studied. Symptoms before and after surgical intervention were compared.Main outcome measures: Pre- vs. post-operative DHI, HIT-6, and audiometric data were compared statistically. The thresholds and amplitudes for cVEMP in symptomatic ears, ears with cochlea-facial nerve dehiscence and ears without CFD were compared statistically.Results: There was a highly significant improvement in DHI and HIT-6 at pre- vs. post-operative (p < 0.0001 and p < 0.001, respectively). The age range was 12.8–52.9 years at the time of surgery (mean = 24.7 years). There were 6 females and 2 males. All 8 had a history of trauma before the onset of their symptoms. The mean cVEMP threshold was 75 dB nHL (SD 3.8) for the operated ear and 85.7 dB (SD 10.6) for the unoperated ear. In contrast to superior semicircular canal dehiscence, where most ears have abnormal ECoG findings suggestive of endolymphatic hydrops, only 1 of 8 operated CFD ears (1 of 16 ears) had an abnormal ECoG study.Conclusions: Overall there was a marked improvement in DHI, HIT-6 and symptoms post-operatively. Statistically significant reduction in cVEMP thresholds was observed in patients with radiographic evidence of CFD. Surgical management with round window reinforcement in patients with CFD was associated with improved symptoms and outcomes measures.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

This study examined the dynamic coordination between disconjugate, vergence eye movements, and pupil size in 52 normal subjects during binocular disparity stimulation in a virtual reality display. Eye movements and pupil area were sampled with a video-oculographic system at 100 Hz during performance of two tasks, (1) fusion of a binocular disparity step (±1.5° of visual angle in the horizontal plane) and (2) pursuit of a sinusoidally varying binocular disparity stimulus (0.1 Hz, ±2.6° of visual angle in the horizontal plane). Pupil size data were normalized on the basis of responses to homogeneous illumination increments ranging from 0.42 to 65.4 cd/m2. The subjects produced robust vergence eye movements in response to disparity step shifts and high fidelity sinusoidal vergence responses (R2 relative to stimulus profile: 0.933 ± 0.088), accompanied by changes in pupil area. Trajectory plots of pupil area as a function of vergence angle showed that the pupil area at zero vergence is altered between epochs of linear vergence angle—pupil area relations. Analysis with a modified Gath-Geva clustering algorithm revealed that the dynamic relationship between the ocular vergence angle and pupil size includes two different transient, synkinetic response patterns. The near response pattern, pupil constriction during convergence and pupil dilation during divergence, occurred ~80% of the time across subjects. An opposite, previously undescribed synkinetic pattern was pupil constriction during divergence and pupil dilatation during convergence; it occurred ~15% of the time across subjects. The remainder of the data were epochs of uncorrelated activity. The pupil size intercepts of the synkinetic segments, representing pupil size at initial tropia, had different relationships to vergence angle for the two main coordinated movement types. Hippus-like movements of the pupil could also be accompanied by vergence movements. No pupil coordination was observed during a conjugate pursuit task. In terms of the current dual interaction control model (1), findings suggest that the synkinetic eye and pupillary movements are produced by a dynamic switch of the influence of vergence related information to pupil control, accompanied by a resetting of the pupil aperture size at zero-vergence.

Most bacteria in nature live in surface-associated communities rather than planktonic populations. Nonetheless, how surface-associated environments shape bacterial evolutionary adaptation remains poorly understood. Here, we show that subjecting Pseudomonas aeruginosa to repeated rounds of swarming, a collective form of surface migration, drives remarkable parallel evolution toward a hyperswarmer phenotype. In all independently evolved hyperswarmers, the reproducible hyperswarming phenotype is caused by parallel point mutations in a flagellar synthesis regulator, FleN, which locks the naturally monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although hyperswarmers outcompete the ancestral strain in swarming competitions, they are strongly outcompeted in biofilm formation, which is an essential trait for P. aeruginosa in environmental and clinical settings. The finding that evolution in swarming colonies reliably produces evolution of poor biofilm formers supports the existence of an evolutionary trade-off between motility and biofilm formation.

ABSTRACT Skin and soft tissue infections (SSTIs) are common in the general population, with increased prevalence among military trainees. Previous research has revealed numerous nasal microbial signatures that correlate with SSTI development and Staphylococcus aureus colonization. Thus, we hypothesized that the ecology of the inguinal, oropharynx, and perianal regions may also be altered in response to SSTI and/or S. aureus colonization. We collected body site samples from 46 military trainees with purulent abscess (SSTI group) as well as from 66 asymptomatic controls (non-SSTI group). We also collected abscess cavity samples to assess the microbial composition of these infections. Samples were analyzed by culture, and the microbial communities were characterized by high-throughput sequencing. We found that the nasal, inguinal, and perianal regions were similar in microbial composition and significantly differed from the oropharynx. We also observed differences in Anaerococcus and Streptococcus abundance between the SSTI and non-SSTI groups for the nasal and oropharyngeal regions, respectively. Furthermore, we detected community membership differences between the SSTI and non-SSTI groups for the nasal and inguinal sites. Compared to that of the other regions, the microbial compositions of the nares of S. aureus carriers and noncarriers were dramatically different; we noted an inverse correlation between the presence of Corynebacterium and the presence of Staphylococcus in the nares. This correlation was also observed for the inguinal region. Culture analysis revealed elevated methicillin-resistant S. aureus (MRSA) colonization levels for the SSTI group in the nasal and inguinal body sites. Together, these data suggest significant microbial variability in patients with SSTI as well as between S. aureus carriers and noncarriers. IMPORTANCE While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs.

The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatusbafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli.

Encounters among bacteria and their viral predators (bacteriophages) are among the most common ecological interactions on Earth. These encounters are likely to occur with regularity inside surface-bound communities that microbes most often occupy in natural environments. Such communities, termed biofilms, are spatially constrained: interactions become limited to near neighbors, diffusion of solutes and particulates can be reduced, and there is pronounced heterogeneity in nutrient access and physiological state. It is appreciated from prior theoretical work that phage-bacteria interactions are fundamentally different in spatially structured contexts, as opposed to well-mixed liquid culture. Spatially structured communities are predicted to promote the protection of susceptible host cells from phage exposure, and thus weaken selection for phage resistance. The details and generality of this prediction in realistic biofilm environments, however, are not known. Here, we explore phage-host interactions using experiments and simulations that are tuned to represent the essential elements of biofilm communities. Our simulations show that in biofilms, phage-resistant cells-as their relative abundance increases-can protect clusters of susceptible cells from phage exposure, promoting the coexistence of susceptible and phage-resistant bacteria under a large array of conditions. We characterize the population dynamics underlying this coexistence, and we show that coexistence is recapitulated in an experimental model of biofilm growth measured with confocal microscopy. Our results provide a clear view into the dynamics of phage resistance in biofilms with single-cell resolution of the underlying cell-virion interactions, linking the predictions of canonical theory to realistic models and in vitro experiments of biofilm growth.IMPORTANCE In the natural environment, bacteria most often live in communities bound to one another by secreted adhesives. These communities, or biofilms, play a central role in biogeochemical cycling, microbiome functioning, wastewater treatment, and disease. Wherever there are bacteria, there are also viruses that attack them, called phages. Interactions between bacteria and phages are likely to occur ubiquitously in biofilms. We show here, using simulations and experiments, that biofilms will in most conditions allow phage-susceptible bacteria to be protected from phage exposure, if they are growing alongside other cells that are phage resistant. This result has implications for the fundamental ecology of phage-bacteria interactions, as well as the development of phage-based antimicrobial therapeutics.

The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger. In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. Yet, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill-defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically-oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus. Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatus bafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings. Importance The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. f umigatus . Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in Aspergilli.

Microbial populations form intricate macroscopic colonies with diverse morphologies whose functions remain to be fully understood. Despite fungal colonies isolated from environmental and clinical samples revealing abundant intraspecies morphological diversity, it is unclear how this diversity affects fungal fitness and disease progression. Here we observe a notable effect of oxygen tension on the macroscopic and biofilm morphotypes of the human fungal pathogen Aspergillus fumigatus. A hypoxia-typic morphotype is generated through the expression of a subtelomeric gene cluster containing genes that alter the hyphal surface and perturb interhyphal interactions to disrupt in vivo biofilm and infection site morphologies. Consequently, this morphotype leads to increased host inflammation, rapid disease progression and mortality in a murine model of invasive aspergillosis. Taken together, these data suggest that filamentous fungal biofilm morphology affects fungal-host interactions and should be taken into consideration when assessing virulence and host disease progression of an isolated strain.

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. It is commonly assumed that biofilm cells are glued together by the matrix; however, how the specific biochemistry of matrix components affects the cell-matrix interactions and how these interactions vary during biofilm growth remain unclear. Here, we investigate cell-matrix interactions in Vibrio cholerae, the causative agent of cholera. We combine genetics, microscopy, simulations, and biochemical analyses to show that V. cholerae cells are not attracted to the main matrix component (Vibrio polysaccharide, VPS), but can be attached to each other and to the VPS network through surface-associated VPS and crosslinks formed by the protein Bap1. Downregulation of VPS production and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results shed light on the dynamics of diverse cell-matrix interactions as drivers of biofilm development. It is commonly assumed that bacterial cells within biofilms are glued together by matrix components, but the details are poorly understood. Here, Moreau et al. show how dynamic changes in attractive and repulsive interactions between cells and various matrix components drive biofilm growth and disassembly in Vibrio cholerae. [ABSTRACT FROM AUTHOR]

Bacteria sense population density via the cell–cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell–cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in which Vibrio cells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically "locked" at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating-mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild type, but as glucose is depleted, wild type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants. Vibrio bacteria sense their surroundings, such as changes in cell density and nutrients, and respond to them using various signaling pathways. This study reveals a link between quorum sensing and metabolic regulation that confers fitness advantages in the presence of certain nutrients. [ABSTRACT FROM AUTHOR]

In the wild, bacteria are predominantly associated with surfaces as opposed to existing as free-swimming, isolated organisms. They are thus subject to surface-specific mechanics, including hydrodynamic forces, adhesive forces, the rheology of their surroundings, and transport rules that define their encounters with nutrients and signaling molecules. Here, we highlight the effects of mechanics on bacterial behaviors on surfaces at multiple length scales, from single bacteria to the development of multicellular bacterial communities such as biofilms.