Showing papers in "Mbio in 2014"

Revealing the Bacterial Butyrate Synthesis Pathways by Analyzing (Meta)genomic Data

Abstract: Butyrate-producing bacteria have recently gained attention, since they are important for a healthy colon and when altered contribute to emerging diseases, such as ulcerative colitis and type II diabetes. This guild is polyphyletic and cannot be accurately detected by 16S rRNA gene sequencing. Consequently, approaches targeting the terminal genes of the main butyrate- producing pathway have been developed. However, since additional pathways exist and alternative, newly recognized enzymes catalyzing the terminal reaction have been described, previous investigations are often incomplete. We undertook a broad analy- sis of butyrate-producing pathways and individual genes by screening 3,184 sequenced bacterial genomes from the Integrated Microbial Genome database. Genomes of 225 bacteria with a potential to produce butyrate were identified, including many pre- viously unknown candidates. The majority of candidates belong to distinct families within the Firmicutes, but members of nine other phyla, especially from Actinobacteria,Bacteroidetes,Fusobacteria,Proteobacteria,Spirochaetes, andThermotogae, were also identified as potential butyrate producers. The established gene catalogue (3,055 entries) was used to screen for butyrate synthesis pathways in 15 metagenomes derived from stool samples of healthy individuals provided by the HMP (Human Micro- biome Project) consortium. A high percentage of total genomes exhibited a butyrate-producing pathway (mean, 19.1%; range, 3.2% to 39.4%), where the acetyl-coenzyme A (CoA) pathway was the most prevalent (mean, 79.7% of all pathways), followed by the lysine pathway (mean, 11.2%). Diversity analysis for the acetyl-CoA pathway showed that the same fewfirmicute groups as- sociated with several Lachnospiraceae andRuminococcaceae were dominating in most individuals, whereas the other pathways were associated primarily with Bacteroidetes. IMPORTANCE Microbiome research has revealed new, important roles of our gut microbiota for maintaining health, but an un- derstanding of effects of specific microbial functions on the host is in its infancy, partly because in-depth functional microbial analyses are rare and publicly available databases are often incomplete/misannotated. In this study, we focused on production of butyrate, the main energy source for colonocytes, which plays a critical role in health and disease. We have provided a complete database of genes from major known butyrate-producing pathways, using in-depth genomic analysis of publicly available ge- nomes,filling an important gap to accurately assess the butyrate-producing potential of complex microbial communities from "-omics"-derived data. Furthermore, a reference data set containing the abundance and diversity of butyrate synthesis pathways from the healthy gut microbiota was established through a metagenomics-based assessment. This study will help in understand- ing the role of butyrate producers in health and disease and may assist the development of treatments for functional dysbiosis.

Conditionally Rare Taxa Disproportionately Contribute to Temporal Changes in Microbial Diversity

Abstract: Microbial communities typically contain many rare taxa that make up the majority of the observed membership, yet the contribution of this microbial "rare biosphere" to community dynamics is unclear. Using 16S rRNA amplicon sequencing of 3,237 samples from 42 time series of microbial communities from nine different ecosystems (air; marine; lake; stream; adult hu- man skin, tongue, and gut; infant gut; and brewery wastewater treatment), we introduce a new method to detect typically rare microbial taxa that occasionally become very abundant (conditionally rare taxa (CRT)) and then quantify their contributions to temporal shifts in community structure. We discovered that CRT made up 1.5 to 28% of the community membership, repre- sented a broad diversity of bacterial and archaeal lineages, and explained large amounts of temporal community dissimilarity (i.e., up to 97% of Bray-Curtis dissimilarity). Most of the CRT were detected at multiple time points, though we also identified "one-hit wonder" CRT that were observed at only one time point. Using a case study from a temperate lake, we gained additional insights into the ecology of CRT by comparing routine community time series to large disturbance events. Our results reveal that many rare taxa contribute a greater amount to microbial community dynamics than is apparent from their low proportional abundances. This observation was true across a wide range of ecosystems, indicating that these rare taxa are essential for under- standing community changes over time. IMPORTANCE Microbial communities and their processes are the foundations of ecosystems. The ecological roles of rare microor- ganisms are largely unknown, but it is thought that they contribute to community stability by acting as a reservoir that can rap- idly respond to environmental changes. We investigated the occurrence of typically rare taxa that very occasionally become more prominent in their communities ("conditionally rare"). We quantified conditionally rare taxa in time series from a wide variety of ecosystems and discovered that not only were conditionally rare taxa present in all of the examples, but they also contributed disproportionately to temporal changes in diversity when they were most abundant. This result indicates an important and gen- eral role for rare microbial taxa within their communities.

The Female Urinary Microbiome: a Comparison of Women with and without Urgency Urinary Incontinence

Abstract: Bacterial DNA and live bacteria have been detected in human urine in the absence of clinical infection, challenging the prevailing dogma that urine is normally sterile. Urgency urinary incontinence (UUI) is a poorly understood urinary condition characterized by symptoms that overlap urinary infection, including urinary urgency and increased frequency with urinary incontinence. The recent discovery of the urinary microbiome warrants investigation into whether bacteria contribute to UUI. In this study, we used 16S rRNA gene sequencing to classify bacterial DNA and expanded quantitative urine culture (EQUC) techniques to isolate live bacteria in urine collected by using a transurethral catheter from women with UUI and, in comparison, a cohort without UUI. For these cohorts, we demonstrated that the UUI and non-UUI urinary microbiomes differ by group based on both sequence and culture evidences. Compared to the non-UUI microbiome, sequencing experiments revealed that the UUI microbiome was composed of increased Gardnerella and decreased Lactobacillus . Nine genera ( Actinobaculum , Actinomyces , Aerococcus , Arthrobacter , Corynebacterium , Gardnerella , Oligella , Staphylococcus , and Streptococcus ) were more frequently cultured from the UUI cohort. Although Lactobacillus was isolated from both cohorts, distinctions existed at the species level, with Lactobacillus gasseri detected more frequently in the UUI cohort and Lactobacillus crispatus most frequently detected in controls. Combined, these data suggest that potentially important differences exist in the urinary microbiomes of women with and without UUI, which have strong implications in prevention, diagnosis, or treatment of UUI. IMPORTANCE New evidence indicates that the human urinary tract contains microbial communities; however, the role of these communities in urinary health remains to be elucidated. Urgency urinary incontinence (UUI) is a highly prevalent yet poorly understood urinary condition characterized by urgency, frequency, and urinary incontinence. Given the significant overlap of UUI symptoms with those of urinary tract infections, it is possible that UUI may have a microbial component. We compared the urinary microbiomes of women affected by UUI to those of a comparison group without UUI, using both high-throughput sequencing and extended culture techniques. We identified statistically significant differences in the frequency and abundance of bacteria present. These differences suggest a potential role for the urinary microbiome in female urinary health.

Middle East Respiratory Syndrome Coronavirus Infection in Dromedary Camels in Saudi Arabia

Abstract: The Middle East respiratory syndrome (MERS) is proposed to be a zoonotic disease; however, the reservoir and mechanism for transmission of the causative agent, the MERS coronavirus, are unknown. Dromedary camels have been implicated through reports that some victims have been exposed to camels, camels in areas where the disease has emerged have antibodies to the virus, and viral sequences have been recovered from camels in association with outbreaks of the disease among humans. Nonetheless, whether camels mediate transmission to humans is unresolved. Here we provide evidence from a geographic and temporal survey of camels in the Kingdom of Saudi Arabia that MERS coronaviruses have been circulating in camels since at least 1992, are distributed countrywide, and can be phylogenetically classified into clades that correlate with outbreaks of the disease among humans. We found no evidence of infection in domestic sheep or domestic goats. IMPORTANCE This study was undertaken to determine the historical and current prevalence of Middle East respiratory syndrome (MERS) coronavirus infection in dromedary camels and other livestock in the Kingdom of Saudi Arabia, where the index case and the majority of cases of MERS have been reported.

Intestinal Dysbiosis Associated with Systemic Lupus Erythematosus

Abstract: Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease in humans and is characterized by the presence of hyperactive immune cells and aberrant antibody responses to nuclear and cytoplasmic antigens, including characteristic anti–double-stranded DNA antibodies. We performed a cross-sectional study in order to determine if an SLE-associated gut dysbiosis exists in patients without active disease. A group of 20 SLE patients in remission, for which there was strict inclusion and exclusion criteria, was recruited, and we used an optimized Ion Torrent 16S rRNA gene-based analysis protocol to decipher the fecal microbial profiles of these patients and compare them with those of 20 age- and sex-matched healthy control subjects. We found diversity to be comparable based on Shannon’s index. However, we saw a significantly lower Firmicutes / Bacteroidetes ratio in SLE individuals (median ratio, 1.97) than in healthy subjects (median ratio, 4.86; P Firmicutes / Bacteroidetes ratio in SLE individuals was corroborated by quantitative PCR analysis. Notably, a decrease of some Firmicutes families was also detected. This dysbiosis is reflected, based on in silico functional inference, in an overrepresentation of oxidative phosphorylation and glycan utilization pathways in SLE patient microbiota. IMPORTANCE Growing evidence suggests that the gut microbiota might impact symptoms and progression of some autoimmune diseases. However, how and why this microbial community influences SLE remains to be elucidated. This is the first report describing an SLE-associated intestinal dysbiosis, and it contributes to the understanding of the interplay between the intestinal microbiota and the host in autoimmune disorders.

Selection of a Multidrug Resistance Plasmid by Sublethal Levels of Antibiotics and Heavy Metals

Abstract: How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments. Importance: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to biocides and heavy metals. In this report, we show that very low concentrations of single antibiotics and heavy metals or combinations of compounds can select for a large plasmid that carries resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic. Our findings suggest that the low levels of antibiotics and heavy metals present in polluted external environments and in treated animals and humans could allow for selection and enrichment of bacteria with multiresistance plasmids and thereby contribute to the emergence, maintenance, and transmission of antibiotic-resistant disease-causing bacteria.

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

Abstract: Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. However, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to such approaches. Unfortunately, such common lab isolates have also been passaged frequently in vitro and have thus lost many of the attributes of wild isolates, often affecting important traits, like virulence. On the other hand, wild isolates are often not amenable to standard genetic approaches, thus limiting inquiry about the genetic basis of biological diversity. Here we imported a new genetic system based on CRISPR/CAS9, which allows high efficiency of targeted gene disruption in natural isolates of T. gondii. This advance promises to bring the power of genetics to bear on the broad diversity of T. gondii strains that have been described recently.

A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students

Abstract: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students’ interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

Microbiota-Induced Changes in Drosophila melanogaster Host Gene Expression and Gut Morphology

Abstract: To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the geneti- cally tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of singleflies revealed high interfly variability that correlated with differences in feeding. To under- stand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally rearedflies to that for their axenically reared counterparts. Our analysis of two wild-typefly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and oldflies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in olderflies. In addition, comparison of wild-type gene expression with that of an immune- deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given thesefindings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far- reaching effects on host physiology. IMPORTANCE The guts of animals are in constant association with microbes, and these interactions are understood to have im- portant roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruitfly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well- established and genetically tractable invertebrate model.

Differential Modulation by Akkermansia muciniphila and Faecalibacterium prausnitzii of Host Peripheral Lipid Metabolism and Histone Acetylation in Mouse Gut Organoids

Abstract: The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithe- lium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkerman- sia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propi- onate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Ppar), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illus- trates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We dem- onstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. IMPORTANCE We investigated the influence of the gut microbiota and microbially produced short-chain fatty acids (SCFAs) on gut functioning. Many commensal bacteria in the gut produce SCFAs, particularly butyrate, acetate, and propionate, which have been demonstrated to reduce the risk of gastrointestinal disorders. Organoids—small crypt-villus structures grown from ileal intestinal stem cells—were exposed to SCFAs and two specific gut bacteria. Akkermansia muciniphila, found in the intestinal mucus, was recently shown to have a favorable effect on the disrupted metabolism associated with obesity. Faecalibacterium prausnitzii is a commensal gut bacterium, the absence of which may be associated with Crohn's disease. We showed that in our model, A. muciniphila induces stronger effects on the host than F. prausnitzii. We observed that A. muciniphila and propionate affect the expression of genes involved in host lipid metabolism and epigenetic activation or silencing of gene expression. We demonstrated that organoids provide a powerful tool for host-microbe interaction studies.

Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems.

Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. IMPORTANCE Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions, what remains elusive is a generalized and programmable strategy that can distinguish between even closely related microorganisms and that allows for fine control over the composition of a microbial population. This study demonstrates that RNA-directed immune systems in bacteria and archaea called CRISPR-Cas systems can provide such a strategy. These systems can be employed to selectively and quantitatively remove individual bacterial strains based purely on sequence information, creating opportunities in the treatment of multidrug-resistant infections, the control of industrial fermentations, and the study of microbial consortia.

Metatranscriptomics of the Human Oral Microbiome during Health and Disease

Abstract: The human microbiome plays important roles in health, but when disrupted, these same indigenous microbes can cause disease. The composition of the microbiome changes during the transition from health to disease; however, these changes are often not conserved among patients. Since microbiome-associated diseases like periodontitis cause similar patient symptoms despite interpatient variability in microbial community composition, we hypothesized that human-associated microbial communities undergo conserved changes in metabolism during disease. Here, we used patient-matched healthy and diseased samples to compare gene expression of 160,000 genes in healthy and diseased periodontal communities. We show that health- and disease-associated communities exhibit defined differences in metabolism that are conserved between patients. In contrast, the metabolic gene expression of individual species was highly variable between patients. These results demonstrate that despite high interpatient variability in microbial composition, disease-associated communities display conserved metabolic profiles that are generally accomplished by a patient-specific cohort of microbes. IMPORTANCE The human microbiome project has shown that shifts in our microbiota are associated with many diseases, including obesity, Crohn's disease, diabetes, and periodontitis. While changes in microbial populations are apparent during these diseases, the species associated with each disease can vary from patient to patient. Taking into account this interpatient variability, we hypothesized that specific microbiota-associated diseases would be marked by conserved microbial community behaviors. Here, we use gene expression analyses of patient-matched healthy and diseased human periodontal plaque to show that microbial communities have highly conserved metabolic gene expression profiles, whereas individual species within the community do not. Furthermore, disease-associated communities exhibit conserved changes in metabolic and virulence gene expression.

Detection of Zoonotic Pathogens and Characterization of Novel Viruses Carried by Commensal Rattus norvegicus in New York City

Abstract: Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoonotic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted molecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, including sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. The two novel hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. Importance: The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide-scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human populations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats collected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Our findings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for increased surveillance and awareness of the disease risks associated with urban rodent infestation.

Characterization of a Novel Influenza Virus in Cattle and Swine: Proposal for a New Genus in the Orthomyxoviridae Family

Abstract: We have recently reported the isolation of a novel virus, provisionally designated C/swine/Oklahoma/1334/2011 (C/OK), with 50% overall homology to human influenza C viruses (ICV), from a pig in Oklahoma. Deep RNA sequencing of C/OK virus found a matrix 1 (M1) protein expression strategy that differed from that of ICV. The novelty of C/OK virus prompted us to investigate whether C/OK virus could exist in a nonswine species. Significantly, we found that C/OK virus was widespread in U.S. bovine herds, as demonstrated by reverse transcription (RT)-PCR and serological assays. Genome sequencing of three bovine viruses isolated from two herds in different states further confirmed these findings. To determine whether swine/bovine C/OK viruses can undergo reassortment with human ICV, and to clarify the taxonomic status of C/OK, in vitro reassortment and serological typing by agar gel immunodiffusion (AGID) were conducted. In vitro reassortment using two human ICV and two swine and bovine C/OK viruses demonstrated that human ICV and C/OK viruses were unable to reassort and produce viable progeny. Antigenically, no cross-recognition of detergent split virions was observed in AGID between human and nonhuman viruses by using polyclonal antibodies that were reactive to cognate antigens. Taken together, these results demonstrate that C/OK virus is genetically and antigenically distinct from ICV. The classification of the new virus in a separate genus of the Orthomyxoviridae family is proposed. The finding of C/OK virus in swine and bovine indicates that this new virus may spread and establish infection in other mammals, including humans. IMPORTANCE Influenza C viruses (ICV) are common human pathogens, infecting most people during childhood and adolescence, and typically cause mild respiratory symptoms. While ICV have been isolated from both pigs and dogs, humans are thought to be the natural viral reservoir. Previously, we characterized an ICV-like virus isolated from pigs exhibiting symptoms of influenza virus-like illness. Here, we show molecular and serological data demonstrating widespread circulation of similar viruses in bovines. Deep RNA sequencing, phylogenetic analysis, and in vitro reassortment experiments demonstrate that animal ICV-like viruses are genetically distinct from human ICV. Antigenically, we show that ICV-like viruses are not recognized by ICV antibodies. En masse, these results suggest that bovine influenza virus warrants classification as a new genus of influenza virus. The finding of this novel virus that can infect multiple mammalian species warrants further research into its role in human health.

Recovery of the Gut Microbiome following Fecal Microbiota Transplantation

Abstract: Clostridium difficile infection is one of the most common health care-associated infections, and up to 40% of patients suffer from recurrence of disease following standard antibiotic therapy. Recently, fecal microbiota transplantation (FMT) has been successfully used to treat recurrent C. difficile infection. It is hypothesized that FMT aids in recovery of a microbiota capa- ble of colonization resistance to C. difficile. However, it is not fully understood how this occurs. Here we investigated changes in the fecal microbiota structure following FMT in patients with recurrent C. difficile infection, and imputed a hypothetical func- tional profile based on the 16S rRNA profile using a predictive metagenomic tool. Increased relative abundance of Bacteroidetes and decreased abundance of Proteobacteria were observed following FMT. The fecal microbiota of recipients following trans- plantation was more diverse and more similar to the donor profile than the microbiota prior to transplantation. Additionally, we observed differences in the imputed metagenomic profile. In particular, amino acid transport systems were overrepresented in samples collected prior to transplantation. These results suggest that functional changes accompany microbial structural changes following this therapy. Further identification of the specific community members and functions that promote coloniza- tion resistance may aid in the development of improved treatment methods for C. difficile infection. IMPORTANCE Within the last decade, Clostridium difficile infection has surpassed other bacterial infections to become the lead- ing cause of nosocomial infections. Antibiotic use, which disrupts the gut microbiota and its capability in providing colonization resistance against C. difficile, is a known risk factor in C. difficile infection. In particular, recurrent C. difficile remains difficult to treat with standard antibiotic therapy. Fecal microbiota transplantation (FMT) has provided a successful treatment method for some patients with recurrent C. difficile infection, but its mechanism and long-term effects remain unknown. Our results provide insight into the structural and potential metabolic changes that occur following FMT, which may aid in the development of new treatment methods for C. difficile infection.

Microbiome Data Distinguish Patients with Clostridium difficile Infection and Non-C. difficile-Associated Diarrhea from Healthy Controls

Abstract: Antibiotic usage is the most commonly cited risk factor for hospital-acquired Clostridium difficile infections (CDI). The increased risk is due to disruption of the indigenous microbiome and a subsequent decrease in colonization resistance by the perturbed bacterial community; however, the specific changes in the microbiome that lead to increased risk are poorly understood. We developed statistical models that incorporated microbiome data with clinical and demographic data to better understand why individuals develop CDI. The 16S rRNA genes were sequenced from the feces of 338 individuals, including cases, diarrheal controls, and nondiarrheal controls. We modeled CDI and diarrheal status using multiple clinical variables, including age, antibiotic use, antacid use, and other known risk factors using logit regression. This base model was compared to models that incorporated microbiome data, using diversity metrics, community types, or specific bacterial populations, to identify characteristics of the microbiome associated with CDI susceptibility or resistance. The addition of microbiome data significantly improved our ability to distinguish CDI status when comparing cases or diarrheal controls to nondiarrheal controls. However, only when we assigned samples to community types was it possible to differentiate cases from diarrheal controls. Several bacterial species within the Ruminococcaceae, Lachnospiraceae, Bacteroides, and Porphyromonadaceae were largely absent in cases and highly associated with nondiarrheal controls. The improved discriminatory ability of our microbiome-based models confirms the theory that factors affecting the microbiome influence CDI. IMPORTANCE The gut microbiome, composed of the trillions of bacteria residing in the gastrointestinal tract, is responsible for a number of critical functions within the host. These include digestion, immune system stimulation, and colonization resistance. The microbiome's role in colonization resistance, which is the ability to prevent and limit pathogen colonization and growth, is key for protection against Clostridium difficile infections. However, the bacteria that are important for colonization resistance have not yet been elucidated. Using statistical modeling techniques and different representations of the microbiome, we demonstrated that several community types and the loss of several bacterial populations, including Bacteroides, Lachnospiraceae, and Ruminococcaceae, are associated with CDI. Our results emphasize the importance of considering the microbiome in mediating colonization resistance and may also direct the design of future multispecies probiotic therapies.

Membership and Behavior of Ultra-Low-Diversity Pathogen Communities Present in the Gut of Humans during Prolonged Critical Illness

Abstract: We analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a “commensal lifestyle.” However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style. IMPORTANCE During critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis.

Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens

Abstract: The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) repre- sent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon con- tact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative patho- gens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a4t o 5l og reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caeno- rhabditis elegans). IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that can- not be treated byfirst-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endoly- sins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

Spread, Circulation, and Evolution of the Middle East Respiratory Syndrome Coronavirus

Abstract: The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections. Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10 −3 substitutions per site per year (95% credible interval [95% CI], 8.76 × 10 −4 ; 1.37 × 10 −3 ) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012). Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection. Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number ( R 0 ) for the virus that is less than 1. IMPORTANCE MERS-CoV adaptation toward higher rates of sustained human-to-human transmission appears not to have occurred yet. While MERS-CoV transmission currently appears weak, careful monitoring of changes in MERS-CoV genomes and of the MERS epidemic should be maintained. The observation of phylogenetically related MERS-CoV in geographically diverse locations must be taken into account in efforts to identify the animal source and transmission of the virus.

Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

Abstract: The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications. IMPORTANCE This work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis.

Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination

Abstract: Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

Abstract: CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

Movement of IS26-Associated Antibiotic Resistance Genes Occurs via a Translocatable Unit That Includes a Single IS26 and Preferentially Inserts Adjacent to Another IS26

Abstract: The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma gondii

Abstract: Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. IMPORTANCE Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.

Campylobacter jejuni Is Not Merely a Commensal in Commercial Broiler Chickens and Affects Bird Welfare

Abstract: Campylobacter jejuni is the leading cause of bacterial food-borne infection; chicken meat is its main source. C. jejuni is considered commensal in chickens based on experimental models unrepresentative of commercial production. Here we show that the paradigm of Campylobacter commensalism in the chicken is flawed. Through experimental infection of four commercial breeds of broiler chickens, we show that breed has a significant effect on C. jejuni infection and the immune response of the animals, although these factors have limited impact on the number of bacteria in chicken ceca. All breeds mounted an innate immune response. In some breeds, this response declined when interleukin-10 was expressed, consistent with regulation of the intestinal inflammatory response, and these birds remained healthy. In another breed, there was a prolonged inflammatory response, evidence of damage to gut mucosa, and diarrhea. We show that bird type has a major impact on infection biology of C. jejuni. In some breeds, infection leads to disease, and the bacterium cannot be considered a harmless commensal. These findings have implications for the welfare of chickens in commercial production where C. jejuni infection is a persistent problem. IMPORTANCECampylobacter jejuni is the most common cause of food-borne bacterial diarrheal disease in the developed world. Chicken is the most common source of infection. C. jejuni infection of chickens had previously not been considered to cause disease, and it was thought that C. jejuni was part of the normal microbiota of birds. In this work, we show that modern rapidly growing chicken breeds used in intensive production systems have a strong inflammatory response to C. jejuni infection that can lead to diarrhea, which, in turn, leads to damage to the feet and legs on the birds due to standing on wet litter. The response and level of disease varied between breeds and is related to regulation of the inflammatory immune response. These findings challenge the paradigm that C. jejuni is a harmless commensal of chickens and that C. jejuni infection may have substantial impact on animal health and welfare in intensive poultry production.

AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments

Abstract: Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn 5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn 7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

Novel Entries in a Fungal Biofilm Matrix Encyclopedia

Abstract: Virulence ofCandidais linked with its ability to form biofilms. Once established, biofilm infections are nearly impos- sible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitroand in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to inter- act physically. Surprisingly, a previously identified polysaccharide of functional importance, -1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included -1,2 branched -1,6-mannans (87%) associated with unbranched -1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Func- tional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of ag- gregate matrix with the antifungalfluconazole, consistent with a role in drug impedance and contribution of multiple matrix components. IMPORTANCE This report is thefirst to decipher the complex and unique macromolecular composition of the Candidabiofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further func- tional investigation of the biofilm matrix, a defining trait of this lifestyle.

Diverse Antibiotic Resistance Genes in Dairy Cow Manure

Abstract: Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

Ecological Succession and Stochastic Variation in the Assembly of Arabidopsis thaliana Phyllosphere Communities

Abstract: Bacteria living on the aerial parts of plants (the phyllosphere) are globally abundant and ecologically significant communities and can have significant effects on their plant hosts. Despite their importance, little is known about the ecological processes that drive phyllosphere dynamics. Here, we describe the development of phyllosphere bacterial communities over time on the model plant Arabidopsis thaliana in a controlled greenhouse environment. We used a large number of replicate plants to identify repeatable dynamics in phyllosphere community assembly and reconstructed assembly history by measuring the composition of the airborne community immigrating to plant leaves. We used more than 260,000 sequences from the v5v6 hypervariable region of the 16S rRNA gene to characterize bacterial community structure on 32 plant and 21 air samples over 73 days. We observed strong, reproducible successional dynamics: phyllosphere communities initially mirrored airborne communities and subsequently converged to a distinct community composition. While the presence or absence of particular taxa in the phyllosphere was conserved across replicates, suggesting strong selection for community composition, the relative abundance of these taxa was highly variable and related to the spatial association of individual plants. Our results suggest that stochastic events in early colonization, coupled with dispersal limitation, generated alternate trajectories of bacterial community assembly within the context of deterministic selection for community membership. IMPORTANCE Commensal bacteria associated with plants help protect their hosts against infection and promote growth. Bacteria associated with plant leaves (the “phyllosphere”) are highly abundant and diverse communities, but we have very limited information about their ecology. Here, we describe the formation of phyllosphere communities on the plant model organism Arabidopsis thaliana. We grew a large number of plants in a greenhouse and measured bacterial diversity in the phyllosphere throughout the Arabidopsis life cycle. We also measured the diversity of airborne microbes landing on leaves. Our findings show that plants develop distinctive phyllosphere bacterial communities drawn from low-abundance air populations, suggesting the plant environment is favorable for particular organisms and not others. However, we also found that the relative abundances of bacteria in the phyllosphere are determined primarily by the physical proximity of individual plants. This suggests that a mixture of selective and random forces shapes phyllosphere communities.

The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages

Abstract: The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms re- mained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans trig- gers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After thefirst 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for mor- phogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild- type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and re- duced exposed 1,3-glucan in hyphae. Our report shows how Candidauses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of py- roptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macro- phage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albi- canshyphae.