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The arms race between (bacterio)phages and their hosts is a recognised hot spot for genome evolution. Indeed, phages and their components have historically paved the way for many molecular biology techniques and biotech applications. Further exploration into their complex lifestyles has revealed that phages are often parasitised by distinct types of hyperparasitic mobile genetic elements. These so-called phage satellites exploit phages to ensure their own propagation and horizontal transfer into new bacterial hosts, and their prevalence and peculiar lifestyle has caught the attention of many researchers. Here, we review the parasite–host dynamics of the known phage satellites, their genomic organisation and their hijacking mechanisms. Finally, we discuss how these elements can be repurposed for diverse biotech applications, kindling a new catalogue of exciting tools for microbiology and synthetic biology.

Antimicrobial resistance (AMR) is an accelerating global threat to modern medicine, yet the nature of AMR in the gut microbiome and how AMR is acquired during early life remain largely unknown. In a cohort of 662 Danish children, we characterized the antibiotic resistance genes (ARGs) acquired during the first year of life and assessed the impacts of diverse environmental exposures on ARG load. Our study reveals a clear bimodal distribution of ARG abundance that is driven by the composition of the gut microbiome, especially E. coli. ARG profiles were significantly affected by various environmental factors. Among these factors, the importance of antibiotics diminished with time since treatment. Finally, ARG load and ARG clusters were also associated with the maturity of the gut microbiome and an asthma-associated bacterial composition. These findings broaden our understanding of AMR in the early life, and have critical implications for efforts to mitigate its spread.

Opportunistic pathogens are associated with several widespread, recalcitrant chronic infections in humans. As the pipeline for new antibiotics thins, virulence management presents an alternative solution to the rising antimicrobial resistance crisis in treating chronic infections. However, the nature of virulence in opportunists is not fully understood. The trade-off hypothesis has been a popular rationalization for the evolution of parasitic virulence since it was first proposed in the early 1980s, but whether it accurately models the evolutionary trajectories of opportunistic pathogens is still uncertain. Here, we tested the evolution of virulence in the human opportunist Pseudomonas aeruginosa PA14 in a murine chronic wound model. We found that in a serial passage experiment where transmission potential is no longer an epidemiological restriction, virulence does not necessarily increase as is predicted by the trade-off hypothesis, and in fact may evolve in different directions. We also assessed P. aeruginosa adaptations to a chronic wound after ten rounds of selection, and found that phenotypic heterogeneity in P. aeruginosa is limited in chronic wounds compared to heterogeneity seen in cystic fibrosis (CF) infections. Using whole genome sequencing, we found that genes coding for virulence factors thought to be crucial in P. aeruginosa pathogenesis acquired mutations during adaptation in chronic wounds. Our findings highlight that (i) current virulence models do not adequately explain the diverging evolutionary trajectories observed during P. aeruginosa chronic wound infection, (ii) P. aeruginosa phenotypic heterogeneity is less extensive in chronic wounds than in CF lungs, (iii) genes involved in P. aeruginosa virulence acquire mutations in chronic wounds, and (iv) similar adaptations are employed by P. aeruginosa both in a chronic wound and CF lung.

Background The hamadryas baboon (Papio hamadryas) is a highly social primate that lives in complex multilevel societies exhibiting a wide range of group behaviors akin to humans. In contrast to the widely studied human microbiome, there is a paucity of information on the host-associated microbiomes of nonhuman primates (NHPs). Here, our goal was to understand the microbial composition throughout different body sites of cohabiting baboons. Results We analyzed 170 oral, oropharyngeal, cervical, uterine, vaginal, nasal and rectal samples from 16 hamadryas baboons via 16S rRNA gene sequencing. Additionally, raw Miseq sequencing data from 1041 comparable publicly available samples from the human oral cavity, gut and vagina were reanalyzed using the same pipeline. We compared the baboon and human microbiome of the oral cavity, gut and vagina, showing that the baboon microbiome is distinct from the human. Baboon cohabitants share similar microbial profiles in their cervix, uterus, vagina, and gut. The oral cavity, gut and vagina shared more bacterial amplicon sequence variants (ASVs) in group living baboons than in humans. The shared ASVs had significantly positive correlations between most body sites, suggesting a potential bacterial exchange throughout the body. No significant differences in gut microbiome composition were detected within the maternity line and between maternity lines, suggesting that the offspring gut microbiota is shaped primarily through bacterial exchange among cohabitants. Finally, Lactobacillus was not so predominant in baboon vagina as in the human vagina but was the most abundant genus in the baboon gut. Conclusions This study is the first to provide comprehensive analyses of the baboon microbiota across different body sites. We contrast this to human body sites and find substantially different microbiomes. This group of cohabitating baboons generally showed higher microbial diversity and remarkable similarities between body sites than were observed in humans. These data and findings from one group of baboons can form the basis of future microbiome studies in baboons and be used as a reference in research where the microbiome is expected to impact human modeling with baboons.

Microbes are faced with environmental fluctuations and it is therefore important to understand how change affects bacterial evolution and population dynamics. Here, we evolved a model bacterium, Pseudomonas aeruginosa PA14, under both constant and changing conditions where settlement by biofilm formation or migration by swimming motility was induced. In changing environments, a heterogeneous population evolved with both specialists and generalists. Interestingly, over time, generalists were outcompeted despite being better at both phenotypes compared to the ancestor. However, generalists became dominant when faced with local competition after resettlement. Reintroduction into a burn wound model, the environment PA14 originally was isolated from, further suggested a tradeoff between niche breadth and peak fitness in agreement with the evolution experiments. Mutations in the c-di-GMP system were key for adaptation of the phenotypes. No mutants lost their ability to regulate c-di-GMP, but generalists acquired mutations that optimized their phenotypic response by efficiently adjusting c-di-GMP levels.

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

Background From early life, children are exposed to a multitude of environmental exposures, which may be of crucial importance for healthy development. Here, the environmental microbiota may be of particular interest as it represents the interface between environmental factors and the child. As infants in modern societies spend a considerable amount of time indoors, we hypothesize that the indoor bed dust microbiota might be an important factor for the child and for the early colonization of the airway microbiome. To explore this hypothesis, we analyzed the influence of environmental exposures on 577 dust samples from the beds of infants together with 542 airway samples from the Copenhagen Prospective Studies on Asthma in Childhood2010 cohort. Results Both bacterial and fungal community was profiled from the bed dust. Bacterial and fungal diversity in the bed dust was positively correlated with each other. Bacterial bed dust microbiota was influenced by multiple environmental factors, such as type of home (house or apartment), living environment (rural or urban), sex of siblings, and presence of pets (cat and/or dog), whereas fungal bed dust microbiota was majorly influenced by the type of home (house or apartment) and sampling season. We further observed minor correlation between bed dust and airway microbiota compositions among infants. We also analyzed the transfer of microbiota from bed dust to the airway, but we did not find evidence of transfer of individual taxa. Conclusions Current study explores the influence of environmental factors on bed dust microbiota (both bacterial and fungal) and its correlation with airway microbiota (bacterial) in early life using high-throughput sequencing. Our findings demonstrate that bed dust microbiota is influenced by multiple environmental exposures and could represent an interface between environment and child.

Staphylococcus aureus is a prominent etiological agent of suppurative abscesses. In principle, abscess formation and purulent exudate are classical physiological features of healing and tissue repair. However, S. aureus deploys two coagulases that can usurp this classical host response and form distinct abscess lesions. Here, we establish that during coinfection with coagulase producers and non-producers, coagulases are shared public goods that contribute to staphylococcal persistence, abscess formation, and disease progression. Coagulase-negative mutants that do not produce the public goods themselves are able to exploit those cooperatively secreted by producers and thereby thrive during coinfection at the expense of others. This study shows the importance of social interactions among pathogens concerning clinical outcomes.

The mother’s vaginal microbiota represents the first microbes to which a child is exposed when delivered vaginally. However, little is known about the composition and development of the vaginal microbiota during pregnancy and birth. Here, we analyzed the vaginal microbiota of 57 women in pregnancy week 24, 36 and at birth after rupture of membranes but before delivery, and further compared the composition with that of the gut and airways of the 1-week-old child. The vaginal community structure had dramatic changes in bacterial diversity and taxonomic distribution, yet carried an individual-specific signature. The relative abundance of most bacterial taxa increased stepwise from week 24 of pregnancy until birth, with a gradual decline of Lactobacillus. Mother-to-child vertical transfer, as suggested by sharing, was modest, with the strongest transfer being for Clostridiales followed by Lactobacillales and Enterobacteriales. In conclusion, late gestation is associated with an increase in maternal vaginal microbiota diversity, and vaginal bacteria at birth only modestly predict the composition of the neonatal microbiota.

Summary Antimicrobial resistance (AMR) is an accelerating global threat, yet the nature of AMR in the gut microbiome and how AMR is acquired during early life remain largely unknown. In a cohort of 662 Danish children, we characterized the antibiotic resistance genes (ARGs) acquired during the first year of life and assessed the impacts of diverse environmental exposures on ARG load. Our study reveals a clear bimodal distribution of ARG richness that is driven by the composition of the gut microbiome, especially E. coli. ARG profiles were significantly affected by various environmental factors. Among these factors, the importance of antibiotics diminished with time since treatment. Finally, ARG load and ARG clusters were also associated with the maturity of the gut microbiome and a bacterial composition associated with increased risk of asthma. These findings broaden our understanding of AMR in early life and have critical implications for efforts to mitigate its spread.

Next-Generation Sequencing (NGS) of 16S rRNA gene is now one of the most widely used application to investigate the microbiota at any given body site in research. Since NGS is more sensitive than traditional culture methods (TCMs), many studies have argued for them to replace TCMs. However, are we really ready for this transition? Here we compare the diagnostic efficiency of the two methods using a large number of samples (n = 1,748 fecal and n = 1,790 hypopharyngeal), among healthy children at different time points. Here we show that bacteria identified by NGS represented 75.70% of the unique bacterial species cultured in each sample, while TCM only identified 23.86% of the bacterial species found by amplicon sequencing. We discuss the pros and cons of both methods and provide perspective on how NGS can be implemented effectively in clinical settings., Shashank Gupta, Martin Mortensen et al. compare the diagnostic efficiency of culturing and amplicon sequencing for studying the human microbiome. They show that amplicon sequencing identifies a greater number of species per sample, and can be implemented in clinical settings.

There have been reports of associations between cesarean section delivery and the risk of childhood asthma, potentially mediated through changes in the gut microbiota. We followed 700 children in the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) cohort prospectively from birth. We examined the effects of cesarean section delivery on gut microbial composition by 16S rRNA gene amplicon sequencing during the first year of life. We then explored whether gut microbial perturbations due to delivery mode were associated with a risk of developing asthma in the first 6 years of life. Delivery by cesarean section was accompanied by marked changes in gut microbiota composition at one week and one month of age, but by one year of age only minor differences persisted compared to vaginal delivery. Increased asthma risk was found in children born by cesarean section only if their gut microbiota composition at 1 year of age still retained a cesarean section microbial signature, suggesting that appropriate maturation of the gut microbiota could mitigate against the increased asthma risk associated with gut microbial changes due to cesarean section delivery.

Coagulation is an innate defense mechanism intended to limit blood loss and trap invading pathogens during infection. However, Staphylococcus aureus has the ability to hijack the coagulation cascade and generate clots via secretion of coagulases. Although many S. aureus have this characteristic, some do not. The population dynamics regarding this defining trait have yet to be explored. We report here that coagulases are public goods that confer protection against antimicrobials and immune factors within a local population or community, thus promoting growth and virulence. By utilizing variants of a methicillin-resistant S. aureus we infer that the secretion of coagulases is a cooperative trait, which is subject to exploitation by invading mutants that do not produce the public goods themselves. However, overexploitation, "tragedy of the commons," does not occur at clinically relevant conditions. Our micrographs indicate this is due to spatial segregation and population viscosity. These findings emphasize the critical role of coagulases in a social evolution context and provide a possible explanation as to why the secretion of these public goods is maintained in mixed S. aureus communities.

The increase in antibiotic drug resistance and the low number of new antibacterial drugs approved in the last few decades requires the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are very promising molecules to fight microbial infection since they kill quickly bacteria and, in some cases, target bacterial membrane. Although some AMPs may be stable against proteolytic degradation by chemical modification, in general, low AMP activity and stability in the presence of serum and proteolytic enzymes as well as their cytotoxicity have impaired their clinical translation. Here, we describe a one-step methodology to generate AMP-conjugated gold nanoparticles (Au NPs), with a high concentration of AMPs (CM-SH) (≈240 AMPs per NP), controlled size (14 nm) and low polydispersity. AMP-conjugated Au NPs demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble AMP, as well as low cytotoxicity against human cells. Moreover, the NPs demonstrated high antimicrobial activity after in vivo administration in a chronic wound and in an animal model of systemic infection.

Background Macrolides are commonly prescribed for respiratory infections and asthma-like episodes in children. While their clinical benefits have been proved, concerns regarding the side-effects of their therapeutic use have been raised. Here we assess the short- and long-term impacts of azithromycin on the gut microbiota of young children. Methods We performed a randomized, double-blind, placebo-controlled trial in a group of children aged 12–36 months, diagnosed with recurrent asthma-like symptoms from the COPSAC2010 cohort. Each acute asthma-like episode was randomized to a 3-day course of azithromycin oral solution of 10 mg/kg per day or placebo. Azithromycin reduced episode duration by half, which was the primary end-point and reported previously. The assessment of gut microbiota after treatment was the secondary end-point and reported in this study. Fecal samples were collected 14 days after randomization (N = 59, short-term) and again at age 4 years (N = 49, long-term, of whom N = 18 were placebo treated) and investigated by 16S rRNA gene amplicon sequencing. Findings Short-term, azithromycin caused a 23% reduction in observed richness and 13% reduction in Shannon diversity. Microbiota composition was shifted primarily in the Actinobacteria phylum, especially a reduction of abundance in the genus Bifidobacterium. Long-term (13–39 months after treatment), we did not observe any differences between the azithromycin and placebo recipients in their gut microbiota composition. Interpretation Azithromycin treatment induced a perturbation in the gut microbiota 14 days after randomization but did not have long-lasting effects on the gut microbiota composition. However, it should be noted that our analyses included a limited number of fecal samples for the placebo treated group at age 4 years. Fund Lundbeck Foundation, Danish Ministry of Health, Danish Council for Strategic Research, Capital Region Research Foundation, China Scholarship Council.

The oral pathogen Aggregatibacter actinomycetemcomitans (Aa) resides in infection sites with many microbes, including commensal streptococci such as Streptococcus gordonii (Sg). During infection, Sg promotes the virulence of Aa by producing its preferred carbon source, l-lactate, a phenomenon referred to as cross-feeding. However, as with many streptococci, Sg also produces high levels of the antimicrobial hydrogen peroxide (H2O2), leading to the question of how Aa deals with this potent antimicrobial during coinfection. Here, we show that Aa possesses two complementary responses to H2O2: a detoxification or fight response mediated by catalase (KatA) and a dispersion or flight response mediated by Dispersin B (DspB), an enzyme that dissolves Aa biofilms. Using a murine abscess infection model, we show that both of these responses are required for Sg to promote Aa virulence. Although the role of KatA is to detoxify H2O2 during coinfection, 3D spatial analysis of mixed infections revealed that DspB is required for Aa to spatially organize itself at an optimal distance (>4 µm) from Sg, which we propose allows cross-feeding but reduces exposure to inhibitory levels of H2O2. In addition, these behaviors benefit not only Aa but also Sg, suggesting that fight and flight stimulate the fitness of the community. These results reveal that an antimicrobial produced by a human commensal bacterium enhances the virulence of a pathogenic bacterium by modulating its spatial location in the infection site.

A fundamental aspect of most infectious diseases is the need for the invading microbe to proliferate in the host. However, little is known about the metabolic pathways required for pathogenic microbes to colonize and persist in their hosts. In this study, we used RNA sequencing (RNA-seq) to generate a high-resolution transcriptome of the opportunistic pathogen Aggregatibacter actinomycetemcomitans in vivo . We identified 691 A. actinomycetemcomitans transcriptional start sites and 210 noncoding RNAs during growth in vivo and as a biofilm in vitro . Compared to in vitro biofilm growth on a defined medium, ∼14% of the A. actinomycetemcomitans genes were differentially regulated in vivo . A disproportionate number of genes coding for proteins involved in metabolic pathways were differentially regulated in vivo , suggesting that A. actinomycetemcomitans in vivo metabolism is distinct from in vitro growth. Mutational analyses of differentially regulated genes revealed that formate dehydrogenase H and fumarate reductase are important A. actinomycetemcomitans fitness determinants in vivo . These results not only provide a high-resolution genomic analysis of a bacterial pathogen during in vivo growth but also provide new insight into metabolic pathways required for A. actinomycetemcomitans in vivo fitness.

Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence., Author Summary Soft tissue infections, such as those in burns, bed sores, and diabetic ulcers, are a significant healthcare and economic burden in the developed and developing world. The opportunistic pathogen P. aeruginosa can cause both acute and chronic infections, and the trajectory of these two types of infections is vastly different. We used high-throughput sequencing to profile P. aeruginosa genome-wide gene expression and mutant fitness during mouse model acute and non-diabetic chronic wound infections. Using these data, we show that wounds are nutrient-rich growth environments in which long-chain fatty acids are a primary source of carbon and energy. We also show that the ability to travel along spatio-chemical gradients by chemotaxis is critical for bacterial fitness and virulence in acute but not chronic infections. Our results demonstrate the utility of simultaneous mutant fitness and gene expression profiling to discover critical functions in complex growth environments.

Most infections result from colonization by more than one microbe. Within such polymicrobial infections, microbes often display synergistic interactions that result in increased disease severity. Although many clinical studies have documented the occurrence of synergy in polymicrobial infections, little is known about the underlying molecular mechanisms. A prominent pathogen in many polymicrobial infections is Pseudomonas aeruginosa , a Gram-negative bacterium that displays enhanced virulence during coculture with Gram-positive bacteria. In this study we discovered that during coinfection, P. aeruginosa uses peptidoglycan shed by Gram-positive bacteria as a cue to stimulate production of multiple extracellular factors that possess lytic activity against prokaryotic and eukaryotic cells. Consequently, P. aeruginosa displays enhanced virulence in a Drosophila model of infection when cocultured with Gram-positive bacteria. Inactivation of a gene ( PA0601 ) required for peptidoglycan sensing mitigated this phenotype. Using Drosophila and murine models of infection, we also show that peptidoglycan sensing results in P. aeruginosa -mediated reduction in the Gram-positive flora in the infection site. Our data suggest that P. aeruginosa has evolved a mechanism to survey the microbial community and respond to Gram-positive produced peptidoglycan through production of antimicrobials and toxins that not only modify the composition of the community but also enhance host killing. Additionally, our results suggest that therapeutic strategies targeting Gram-positive bacteria might be a viable approach for reducing the severity of P. aeruginosa polymicrobial infections.

Bacterial growth and virulence often depends upon the cooperative release of extracellular factors excreted in response to quorum sensing (QS). We carried out an in vivo selection experiment in mice to examine how QS evolves in response to variation in relatedness (strain diversity), and the consequences for virulence. We started our experiment with two bacterial strains: a wild-type that both produces and responds to QS signal molecules, and a lasR (signal-blind) mutant that does not release extracellular factors in response to signal. We found that: (i) QS leads to greater growth within hosts; (ii) high relatedness favours the QS wild-type; and (iii) low relatedness favours the lasR mutant. Relatedness matters in our experiment because, at relatively low relatedness, the lasR mutant is able to exploit the extracellular factors produced by the cells that respond to QS, and hence increase in frequency. Furthermore, our results suggest that because a higher relatedness favours cooperative QS, and hence leads to higher growth, this will also lead to a higher virulence, giving a relationship between relatedness and virulence that is in the opposite direction to that usually predicted by virulence theory.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.