The ICU environment contributes to the endemicity of the "Serratia marcescens complex" in the hospital setting.

Serratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019-2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019) with 165 genomic data. These genomes were grouped into clades (1-4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens/Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%-98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC ß-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia. We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying blaVIM-1 or blaOXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both "source" (permanent, sinks) and "sink" (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe "hospital environment," including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals.

The respiratory tract microbiome, the pathogen load, and clinical interventions define severity of bacterial pneumonia.

Bacterial pneumonia is a considerable problem worldwide. Here, we follow the inter-kingdom respiratory tract microbiome (RTM) of a unique cohort of 38 hospitalized patients (n = 97 samples) with pneumonia caused by Legionella pneumophila. The RTM composition is characterized by diversity drops early in hospitalization and ecological species replacement. RTMs with the highest bacterial and fungal loads show low diversity and pathogen enrichment, suggesting high biomass as a biomarker for secondary and/or co-infections. The RTM structure is defined by a "commensal" cluster associated with a healthy RTM and a "pathogen" enriched one, suggesting that the cluster equilibrium drives the microbiome to recovery or dysbiosis. Legionella biomass correlates with disease severity and co-morbidities, while clinical interventions influence the RTM dynamics. Fungi, archaea, and protozoa seem to contribute to progress of pneumonia. Thus, the interplay of the RTM equilibrium, the pathogen load dynamics, and clinical interventions play a critical role in patient recovery.

Ecology of the respiratory tract microbiome.

A thriving multi-kingdom microbial ecosystem inhabits the respiratory tract: the respiratory tract microbiome (RTM). In recent years, the contribution of the RTM to human health has become a crucial research aspect. However, research into the key ecological processes, such as robustness, resilience, and microbial interaction networks, has only recently started. This review leans on an ecological framework to interpret the human RTM and determine how the ecosystem functions and assembles. Specifically, the review illustrates the ecological RTM models and discusses microbiome establishment, community structure, diversity stability, and critical microbial interactions. Lastly, the review outlines the RTM responses to ecological disturbances, as well as the promising approaches for restoring ecological balance.

Antimicrobial Resistance in the Global Health Network: Known Unknowns and Challenges for Efficient Responses in the 21st Century.

Antimicrobial resistance (AMR) is one of the Global Health challenges of the 21st century. The inclusion of AMR on the global map parallels the scientific, technological, and organizational progress of the healthcare system and the socioeconomic changes of the last 100 years. Available knowledge about AMR has mostly come from large healthcare institutions in high-income countries and is scattered in studies across various fields, focused on patient safety (infectious diseases), transmission pathways and pathogen reservoirs (molecular epidemiology), the extent of the problem at a population level (public health), their management and cost (health economics), cultural issues (community psychology), and events associated with historical periods (history of science). However, there is little dialogue between the aspects that facilitate the development, spread, and evolution of AMR and various stakeholders (patients, clinicians, public health professionals, scientists, economic sectors, and funding agencies). This study consists of four complementary sections. The first reviews the socioeconomic factors that have contributed to building the current Global Healthcare system, the scientific framework in which AMR has traditionally been approached in such a system, and the novel scientific and organizational challenges of approaching AMR in the fourth globalization scenario. The second discusses the need to reframe AMR in the current public health and global health contexts. Given that the implementation of policies and guidelines are greatly influenced by AMR information from surveillance systems, in the third section, we review the unit of analysis ("the what" and "the who") and the indicators (the "operational units of surveillance") used in AMR and discuss the factors that affect the validity, reliability, and comparability of the information to be applied in various healthcare (primary, secondary, and tertiary), demographic, and economic contexts (local, regional, global, and inter-sectorial levels). Finally, we discuss the disparities and similarities between distinct stakeholders' objectives and the gaps and challenges of combatting AMR at various levels. In summary, this is a comprehensive but not exhaustive revision of the known unknowns about how to analyze the heterogeneities of hosts, microbes, and hospital patches, the role of surrounding ecosystems, and the challenges they represent for surveillance, antimicrobial stewardship, and infection control programs, which are the traditional cornerstones for controlling AMR in human health.

Sustainable plant-based diets promote rainbow trout gut microbiota richness and do not alter resistance to bacterial infection.

BACKGROUND: Farmed fish food with reduced fish-derived products are gaining growing interest due to the ecological impact of fish-derived protein utilization and the necessity to increase aquaculture sustainability. Although different terrestrial plant proteins could replace fishmeal proteins, their use is associated with adverse effects. Here, we investigated how diets composed of terrestrial vegetal sources supplemented with proteins originating from insect, yeast or terrestrial animal by-products affect rainbow trout (Onchorynchus mykiss) gut microbiota composition, growth performance and resistance to bacterial infection by the fish pathogen Flavobacterium psychrophilum responsible for frequent outbreaks in aquaculture settings. RESULTS: We showed that the tested regimes significantly increased gut bacterial richness compared to full vegetal or commercial-like diets, and that vegetal diet supplemented with insect and yeast proteins improves growth performance compared to full vegetal diet without altering rainbow trout susceptibility to F. psychrophilum infection. CONCLUSION: Our results demonstrate that the use of insect and yeast protein complements to vegetal fish feeds maintain microbiota functions, growth performance and fish health, therefore identifying promising alternative diets to improve aquaculture's sustainability.

Interkingdom Gut Microbiome and Resistome of the Cockroach Blattella germanica.

Cockroaches are intriguing animals with two coexisting symbiotic systems, an endosymbiont in the fat body, involved in nitrogen metabolism, and a gut microbiome whose diversity, complexity, role, and developmental dynamics have not been fully elucidated. In this work, we present a metagenomic approach to study Blattella germanica populations not treated, treated with kanamycin, and recovered after treatment, both naturally and by adding feces to the diet, with the aim of better understanding the structure and function of its gut microbiome along the development as well as the characterization of its resistome.IMPORTANCE For the first time, we analyze the interkingdom hindgut microbiome of this species, including bacteria, fungi, archaea, and viruses. Network analysis reveals putative cooperation between core bacteria that could be key for ecosystem equilibrium. We also show how antibiotic treatments alter microbiota diversity and function, while both features are restored after one untreated generation. Combining data from B. germanica treated with three antibiotics, we have characterized this species' resistome. It includes genes involved in resistance to several broad-spectrum antibiotics frequently used in the clinic. The presence of genetic elements involved in DNA mobilization indicates that they can be transferred among microbiota partners. Therefore, cockroaches can be considered reservoirs of antibiotic resistance genes (ARGs) and potential transmission vectors.

Altered Ecology of the Respiratory Tract Microbiome and Nosocomial Pneumonia.

Nosocomial pneumonia is one of the most frequent infections in critical patients. It is primarily associated with mechanical ventilation leading to severe illness, high mortality, and prolonged hospitalization. The risk of mortality has increased over time due to the rise in multidrug-resistant (MDR) bacterial infections, which represent a global public health threat. Respiratory tract microbiome (RTM) research is growing, and recent studies suggest that a healthy RTM positively stimulates the immune system and, like the gut microbiome, can protect against pathogen infection through colonization resistance (CR). Physiological conditions of critical patients and interventions as antibiotics administration and mechanical ventilation dramatically alter the RTM, leading to dysbiosis. The dysbiosis of the RTM of ICU patients favors the colonization by opportunistic and resistant pathogens that can be part of the microbiota or acquired from the hospital environments (biotic or built ones). Despite recent evidence demonstrating the significance of RTM in nosocomial infections, most of the host-RTM interactions remain unknown. In this context, we present our perspective regarding research in RTM altered ecology in the clinical environment, particularly as a risk for acquisition of nosocomial pneumonia. We also reflect on the gaps in the field and suggest future research directions. Moreover, expected microbiome-based interventions together with the tools to study the RTM highlighting the "omics" approaches are discussed.

Insects' potential: Understanding the functional role of their gut microbiome.

The study of insect-associated microbial communities is a field of great importance in agriculture, principally because of the role insects play as pests. In addition, there is a recent focus on the potential of the insect gut microbiome in areas such as biotechnology, given some microorganisms produce molecules with biotechnological and industrial applications, and also in biomedicine, since some bacteria and fungi are a reservoir of antibiotic resistance genes (ARGs). To date, most studies aiming to characterize the role of the gut microbiome of insects have been based on high-throughput sequencing of the 16S rRNA gene and/or metagenomics. However, recently functional approaches such as metatranscriptomics, metaproteomics and metabolomics have also been employed. Besides providing knowledge about the taxonomic distribution of microbial populations, these techniques also reveal their functional and metabolic capabilities. This information is essential to gain a better understanding of the role played by microbes comprising the microbial communities in their hosts, as well as to indicate their possible exploitation. This review provides an overview of how far we have come in characterizing insect gut functionality through omics, as well as the challenges and future perspectives in this field.

Metagenomic approaches in microbial ecology: an update on whole-genome and marker gene sequencing analyses.

Metagenomics and marker gene approaches, coupled with high-throughput sequencing technologies, have revolutionized the field of microbial ecology. Metagenomics is a culture-independent method that allows the identification and characterization of organisms from all kinds of samples. Whole-genome shotgun sequencing analyses the total DNA of a chosen sample to determine the presence of micro-organisms from all domains of life and their genomic content. Importantly, the whole-genome shotgun sequencing approach reveals the genomic diversity present, but can also give insights into the functional potential of the micro-organisms identified. The marker gene approach is based on the sequencing of a specific gene region. It allows one to describe the microbial composition based on the taxonomic groups present in the sample. It is frequently used to analyse the biodiversity of microbial ecosystems. Despite its importance, the analysis of metagenomic sequencing and marker gene data is quite a challenge. Here we review the primary workflows and software used for both approaches and discuss the current challenges in the field.

Persistent Legionnaires' Disease and Associated Antibiotic Treatment Engender a Highly Disturbed Pulmonary Microbiome Enriched in Opportunistic Microorganisms.

Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections.IMPORTANCE The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance.

Unraveling Assemblage, Functions and Stability of the Gut Microbiota of Blattella germanica by Antibiotic Treatment.

Symbiosis between prokaryotes and eukaryotes is a widespread phenomenon that has contributed to the evolution of eukaryotes. In cockroaches, two types of symbionts coexist: an endosymbiont in the fat body (Blattabacterium), and a rich gut microbiota. The transmission mode of Blattabacterium is vertical, while the gut microbiota of a new generation is mainly formed by bacterial species present in feces. We have carried out a metagenomic analysis of Blattella germanica populations, treated and non-treated with two antibiotics (vancomycin and ampicillin) over two generations to (1) determine the core of bacterial communities and potential functions of the gut microbiota and (2) to gain insights into the mechanisms of resistance and resilience of the gut microbiota. Our results indicate that the composition and functions of the bacteria were affected by treatment, more severely in the case of vancomycin. Further results demonstrated that in an untreated second-generation population that comes from antibiotic-treated first-generation, the microbiota is not yet stabilized at nymphal stages but can fully recover in adults when feces of a control population were added to the diet. This signifies the existence of a stable core in either composition and functions in lab-reared populations. The high microbiota diversity as well as the observed functional redundancy point toward the microbiota of cockroach hindguts as a robust ecosystem that can recover from perturbations, with recovery being faster when feces are added to the diet.

The Gut Microbiota Composition of the Moth Brithys crini Reflects Insect Metamorphosis.

Lepidoptera is a highly diverse insect order with major importance in agriculture as many species are considered pests. The role of the gut microbiota in insect physiology is still poorly understood, despite the research undertaken in recent years. Furthermore, Lepidoptera are holometabolous insects and few studies have addressed the influence of the changes taking place on the gut microbiome composition and diversity during metamorphosis, especially in monophagous species. The V3-V4 region of the 16S rRNA gene was sequenced to investigate the microbiota composition and diversity of the monophagous moth Brithys crini during three different life stages: egg, larvae (midgut and hindgut), and adult (gut). Our results showed that the microbiota composition of B. crini was stage specific, indicating that the developmental stage is a main factor affecting the gut microbiome in composition and potential functions. Moreover, the diversity of the gut microbiome reflected the developmental process, since a drop in diversity occurred between the larval and the adult phase, when the intestine is completely renewed. In spite of the changes in the gut microbiota during metamorphosis, 29 genera were conserved throughout the three developmental stages, mainly belonging to the Proteobacteria phylum, which define the core microbiome of B. crini. These genera seem to contribute to host physiology by participating in food digestion, nutrition, and detoxification mechanisms.

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

Analysis of the Pulmonary Microbiome Composition of Legionella pneumophila-Infected Patients.

The analysis of the lung microbiome composition is a field of research that recently emerged. It gained great interest in pulmonary diseases such as pneumonia since the microbiome seems to be involved in host immune responses, inflammation, and protection against pathogens. Thus, it is possible that the microbial communities living in the lungs play a role in the outcome and severity of lung infections such as Legionella-caused pneumonia and in the response to antibiotic therapy. In this chapter, all steps necessary for the characterization of the bacterial and fungal fraction of the lung microbiome using high-throughput sequencing approaches are explained, starting from the selection of clinical samples to the analysis of the taxonomic composition, diversity, and ecology of the microbiome.

Diet shapes the gut microbiota of the omnivorous cockroach Blattella germanica.

The gut microbiota of insects contributes positively to the physiology of its host mainly by participating in food digestion, protecting against pathogens, or provisioning vitamins or amino acids, but the dynamics of this complex ecosystem is not well understood so far. In this study, we have characterized the gut microbiota of the omnivorous cockroach Blattella germanica by pyrosequencing the hypervariable regions V1-V3 of the 16S rRNA gene of the whole bacterial community. Three diets differing in the protein content (0, 24 and 50%) were tested at two time points in lab-reared individuals. In addition, the gut microbiota of wild adult cockroaches was also analyzed. In contrast to the high microbial richness described on the studied samples, only few species are shared by wild and lab-reared cockroaches, constituting the bacterial core in the gut of B. germanica. Overall, we found that the gut microbiota of B. germanica is highly dynamic as the bacterial composition was reassembled in a diet-specific manner over a short time span, with no-protein diet promoting high diversity, although the highest diversity was found in the wild cockroaches analyzed. We discuss how the flexibility of the gut microbiota is probably due to its omnivorous life style and varied diets.

Colonization Resistance of the Gut Microbiota against Clostridium difficile.

Antibiotics strongly disrupt the human gut microbiota, which in consequence loses its colonization resistance capacity, allowing infection by opportunistic pathogens such as Clostridium difficile. This bacterium is the main cause of antibiotic-associated diarrhea and a current problem in developed countries, since its incidence and severity have increased during the last years. Furthermore, the emergence of antibiotic resistance strains has reduced the efficiency of the standard treatment with antibiotics, leading to a higher rate of relapses. Here, we review recent efforts focused on the impact of antibiotics in the gut microbiome and their relationship with C. difficile colonization, as well as, in the identification of bacteria and mechanisms involved in the protection against C. difficile infection. Since a healthy gut microbiota is able to avoid pathogen colonization, restoration of the gut microbiota seems to be the most promising approach to face C. difficile infection, especially for recurrent cases. Therefore, it would be possible to design probiotics for patients undergoing antimicrobial therapies in order to prevent or fight the expansion of the pathogen in the gut ecosystem.

[Molecular epidemiology studies on the immigrant population in Spain]. / Estudios de epidemiología molecular en población inmigrante en España.

BACKGROUND: Molecular epidemiology is a new scientific discipline which allows to integrate information on the genetic variation of infectious pathogens with their diffusion in a population and its subgroups including, for instance, resistance mutations to antibiotics and antiretrovirals. We present the results of an analysis of scientific publications that analyze the health status of the immigrant population in Spain from a molecular epidemiology perspective. METHODS: We reviewed original articles published in 1998-2014 with the keywords "molecular epidemiology", "molecular typing", "sequencing", "immigrant", and "Spain". RESULTS: From a total of 267 articles identified initially, only 50 passed through the established filters. Most of them (36) analyzed infections by Mycobacterium tuberculosis (3) and HIV (3), followed at a large distance by Staphylococcus aureus and hepatitis B virus. The main goal of these works was the typing of the pathogen and to determine the frequency of resistance mutations. CONCLUSION: Is difficult to generalize the conclusions from the analyzed articles because most of them have a purely descriptive and quite restricted scope, considering the type and size of the samples studied. Several studies are focused on the most likely origin for the strains or variants of the pathogen but others also reveal transmissions from the local to the immigrant populations.

Succession of the gut microbiota in the cockroach Blattella germanica.

The cockroach gut harbors a wide variety of microorganisms that, among other functions, collaborate in digestion and act as a barrier against pathogen colonization. Blattabacterium, a primary endosymbiont, lives in the fat body inside bacteriocytes and plays an important role in nitrogen recycling. Little is known about the mode of acquisition of gut bacteria or their ecological succession throughout the insect life cycle. Here we report on the bacterial taxa isolated from different developmental instars of the cockroach Blattella germanica. The bacterial load in the gut increased two orders of magnitude from the first to the second nymphal stage, coinciding with the incorporation of the majority of bacterial taxa, but remained similar thereafter. Pyrosequencing of the hypervariable regions V1-V3 of the 16S rRNA genes showed that the microbial composition differed significantly between adults and nymphs. Specifically, a succession was observed in which Fusobacterium accumulated with aging, while Bacteroides decreased. Blattabacterium was the only symbiont found in the ootheca, which makes the vertical transmission of gut bacteria an unlikely mode of acquisition. Scanning electron microscopy disclosed a rich bacterial biofilm in third instar nymphs, while filamentous structures were found exclusively in adults.

Differential effects of antibiotic therapy on the structure and function of human gut microbiota.

The human intestinal microbiota performs many essential functions for the host. Antimicrobial agents, such as antibiotics (AB), are also known to disturb microbial community equilibrium, thereby having an impact on human physiology. While an increasing number of studies investigate the effects of AB usage on changes in human gut microbiota biodiversity, its functional effects are still poorly understood. We performed a follow-up study to explore the effect of ABs with different modes of action on human gut microbiota composition and function. Four individuals were treated with different antibiotics and samples were taken before, during and after the AB course for all of them. Changes in the total and in the active (growing) microbiota as well as the functional changes were addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. We have found that the class of antibiotic, particularly its antimicrobial effect and mode of action, played an important role in modulating the gut microbiota composition and function. Furthermore, analysis of the resistome suggested that oscillatory dynamics are not only due to antibiotic-target resistance, but also to fluctuations in the surviving bacterial community. Our results indicated that the effect of AB on the human gut microbiota relates to the interaction of several factors, principally the properties of the antimicrobial agent, and the structure, functions and resistance genes of the microbial community.