Modeling airway persistent infection of Moraxella catarrhalis and nontypeable Haemophilus influenzae by using human in vitro models.

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway in vitro models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks. Notably, the use of these models, coupled with confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets.

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Chronic wound isolates and their minimum inhibitory concentrations against third generation cephalosporins at a tertiary hospital in Uganda.

Globally, the burden of chronic wound infections is likely to increase due to the rising levels of bacterial resistance to antibiotics. In the United States of America alone, more than 6.5 million chronic wounds with evidence of bacterial infection are diagnosed every year. In addition, the polymicrobial environment in chronic wound infections has been observed from several studies as a risk factor for development of resistance to many antibiotics including the third generation cephalosporins currently used in Mbarara Regional Referral Hospital for treatment of chronic wound infections. Therefore the main objective of this study was to determine the prevalence of chronic wound isolates and their minimum inhibitory concentrations (MIC) against third generation cephalosporins. This study was a cross-sectional descriptive and analytical survey of bacterial isolates from chronic wound infection among 75 study participants admitted in the surgical ward of Mbarara Regional Referral Hospital (MRRH), a tertiary Hospital in Western Uganda. Standard laboratory bacterial culture and identification techniques as well as broth microdilution method were used to isolate, identify pathogens and test for MIC respectively. We found that 69/75 study participants had samples with bacterial growth and the most prevalent pathogens isolated were staphylococcus aureus (40.6%) and Klebsiella spp. (29%). Generally, most isolates were susceptible to cefoperazone + sulbactum 2 g (Sulcef) and ceftriaxone 1 g (Epicephin). The overall prevalence of isolates in chronic wound infection among patients admitted in the surgical ward of MRRH was 92% and the most prevalent isolates were Staphylococcus aureus, Klebsiella species and proteus species respectively. The observed MIC values were higher than the CLSI clinical breakpoint, implying a decreasing trend in susceptibility of chronic wound isolates to third generation cephalosporins.

The Role of Integration Host Factor in Escherichia coli Persister Formation.

Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational ihfA-mVenus fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of ihf produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the lac operon, and show that a knockout of the lacI repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions. IMPORTANCE Persisters are phenotypic variants that survive exposure to antibiotics through temporary dormancy. Mutants with increased levels of persisters have been identified in clinical isolates, and evidence suggests these cells contribute to chronic infections and antibiotic treatment failure. Understanding the underlying mechanism of persister formation and tolerance is important for developing therapeutic approaches to treat chronic infections. In this study, we examine a global regulator, IHF, that plays a role in persister formation. We find that noise in expression of IHF contributes to persister formation, likely by regulating the switch between the TCA cycle that efficiently produces energy and the glyoxylate bypass. We extend this study to a simple model lac operon and show that when grown on lactose as the sole carbon source, noise in its expression influences ATP levels and determines persister formation. This noise is quenched by overexpression of the lac operon, providing a simple approach to test the involvement of a gene in persister formation.

Characteristics of the Cervicovaginal Microenvironment in Childbearing-Age Women with Different Degrees of Cervical Lesions and HR-HPV Positivity.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the most important determinate in the development of cervical cancer, and cervical microecology can modulate cervical viral infection. However, few studies have been conducted on the microecological analysis of cervical diseases using strict physiological factors. This study investigated the characteristics and dynamics of cervical microecology in childbearing-age Chinese women with different degrees of HR-HPV-positive cervical lesions. A total of 168 subjects were selected according to the selection criteria, including healthy HPV-negative individuals (n = 29), HR-HPV-infected individuals (n = 29), low-grade squamous intraepithelial lesion individuals (LSIL, n = 32), high-grade squamous intraepithelial lesion individuals (HSIL, n = 40), and cervical cancer individuals (n = 38). We sampled cervical secretions from each subject and performed comparative analysis using the 16S rRNA sequencing method. Comparison analysis showed that Lactobacillus and Ignatzschineria were the dominant genera in the healthy group, while Gardnerella and Prevotella were more enriched in the disease groups. Based on the taxa composition, we roughly divided the development of cervical cancer into two phases: phase I was from healthy status to HR-HPV infection and LSIL; phase II was from LSIL to HSIL and cervical cancer. Different interactions among different genera were observed in different groups. Prevotella inhibited the abundance of Lactobacillus in the healthy group, while Prevotella inhabited the abundance of Gardnerella in the other groups. In the HR-HPV infection group, Ignatzschineria and Enterococcus showed a positive interaction but dissociated with the increase in cervical lesions, which might eventually lead to a continuous decrease in the abundances of Lactobacillus and Ignatzschineria.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections. IMPORTANCE Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are "susceptible" in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these antibiotics frequently fail to eliminate these infections in vivo. Therefore, the persistent outcomes in MRSA infections represent a unique and important variant of classic "antibiotic resistance" that is only disclosed during in vivo antibiotic treatment. Given the high morbidity and mortality associated with the persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between purine biosynthesis and the second messenger c-di-AMP plays an important role in VAN persistence in experimental MRSA endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening infections.

Roles of Two-Component Systems in Pseudomonas aeruginosa Virulence.

Pseudomonas aeruginosa is an opportunistic pathogen that synthesizes and secretes a wide range of virulence factors. P. aeruginosa poses a potential threat to human health worldwide due to its omnipresent nature, robust host accumulation, high virulence, and significant resistance to multiple antibiotics. The pathogenicity of P. aeruginosa, which is associated with acute and chronic infections, is linked with multiple virulence factors and associated secretion systems, such as the ability to form and utilize a biofilm, pili, flagella, alginate, pyocyanin, proteases, and toxins. Two-component systems (TCSs) of P. aeruginosa perform an essential role in controlling virulence factors in response to internal and external stimuli. Therefore, understanding the mechanism of TCSs to perceive and respond to signals from the environment and control the production of virulence factors during infection is essential to understanding the diseases caused by P. aeruginosa infection and further develop new antibiotics to treat this pathogen. This review discusses the important virulence factors of P. aeruginosa and the understanding of their regulation through TCSs by focusing on biofilm, motility, pyocyanin, and cytotoxins.

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Persister cells: formation, resuscitation and combative therapies.

Persister cells, or superfits, have been strongly implicated in the recalcitrance and recurrence of chronic bacterial infection through the dormant (metabolically reduced) phenotype they display and the tolerance to antimicrobial agents this dormancy grants them. The complex biochemical events that lead to the formation of persister cells are not completely understood, though much research has linked the degradation of type II toxin/antitoxin systems and reduced cellular ATP levels to the rise in stress response molecules (where (p)ppGpp is of particular interest), which induce this dormant state. The equally complex mechanism of resuscitation is initiated by the cells' ability to sense nutrient availability via chemotaxis systems. Levels of secondary messenger proteins (i.e., cAMP) within the cell are reduced to allow the resuscitation of ribosomes, by ribosomal resuscitation factor HflX, to reinstate protein synthesis and, therefore, growth to re-populate. Techniques of superfit eradication utilise one, or more, of three approaches (i) direct killing, (ii) re-sensitising persister cells to conventional antimicrobials, or (iii) prevention of persister formation though few laboratory findings have been translated to clinical practice. This work will outline current findings in the field with a critical approach, where possible.

Alkyl-Quinolones derivatives as potential biomarkers for Pseudomonas aeruginosa infection chronicity in Cystic Fibrosis.

In Cystic Fibrosis (CF), a rapid and standardized definition of chronic infection would allow a better management of Pseudomonas aeruginosa (Pa) infections, as well as a quick grouping of patients during clinical trials allowing better comparisons between studies. With this purpose, we compared the metabolic profiles of 44 in vitro cultures of Pa strains isolated from CF patients at different stages of infection in order to identify metabolites differentially synthetized according to these clinical stages. Compounds produced and secreted by each strain in the supernatant of a liquid culture were analysed by metabolomic approaches (UHPLC-DAD-ESI/QTOF, UV and UPLC-Orbitrap, MS). Multivariate analyses showed that first colonization strains could be differentiated from chronic colonization ones, by producing notably more Alkyl-Quinolones (AQs) derivatives. Especially, five AQs were discriminant: HQC5, HQNOC7, HQNOC7:1, db-PQS C9 and HQNOC9:1. However, the production of HHQ was equivalent between strain types. The HHQ/HQNOC9:1 ratio was then found to be significantly different between chronic and primo-colonising strains by using both UV (p = 0.003) and HRMS data (p = 1.5 × 10-5). Our study suggests that some AQ derivatives can be used as biomarkers for an improved management of CF patients as well as a better definition of the clinical stages of Pa infection.

Upper Respiratory Tract Microbiome of Australian Aboriginal and Torres Strait Islander Children in Ear and Nose Health and Disease.

The objective of this study was to examine the nasal microbiota in relation to otitis media (OM) status and nose health in Indigenous Australian children. Children 2 to 7 years of age were recruited from two northern Australian (Queensland) communities. Clinical histories were obtained through parent interviews and reviews of the medical records. Nasal cavity swab samples were obtained, and the children's ears, nose, and throat were examined. DNA was extracted and analyzed by 16S rRNA amplicon next-generation sequencing of the V3/V4 region, in combination with previously generated culture data. A total of 103 children were recruited (mean age, 4.7 years); 17 (16.8%) were healthy, i.e., normal examination results and no history of OM. The nasal microbiota differed significantly in relation to OM status and nose health. Children with historical OM had greater relative abundance of Moraxella, compared to healthy children, despite both having healthy ears at the time of swabbing. Children with healthy noses had greater relative abundance of Staphylococcus aureus, compared to those with rhinorrhea. Dolosigranulum was correlated with Corynebacterium in healthy children. Haemophilus and Streptococcus were correlated across phenotypes. Ornithobacterium was absent or was present with low relative abundance in healthy children and clustered around otopathogens. It correlated with Helcococcus and Dichelobacter. Dolosigranulum and Corynebacterium form a synergism that promotes upper respiratory tract (URT)/ear health in Indigenous Australian children. Ornithobacterium likely represents "Candidatus Ornithobacterium hominis" and in this population is correlated with a novel bacterium that appears to be related to poor URT/ear health. IMPORTANCE Recurring and chronic infections of the ear (OM) are disproportionately prevalent in disadvantaged communities across the globe and, in particular, within Indigenous communities. Despite numerous intervention strategies, OM persists as a major health issue and is the leading cause of preventable hearing loss. In disadvantaged communities, this hearing loss is associated with negative educational and social development outcomes, and consequently, poorer employment prospects and increased contact with the justice system in adulthood. Thus, a better understanding of the microbial ecology is needed in order to identify new targets to treat, as well as to prevent the infections. This study used a powerful combination of 16S rRNA gene sequencing and extended culturomics to show that Dolosigranulum pigrum, a bacterium previously identified as a candidate protective species, may require cocolonization with Corynebacterium pseudodiphtheriticum in order to prevent OM. Additionally, emerging and potentially novel pathogens and bacteria were identified.

Revisiting tuberculosis screening: An insight to complementary diagnosis and prospective molecular approaches for the recognition of the dormant TB infection in human and cattle hosts.

Tuberculosis (TB) is defined as a chronic infection in both human and cattle hosts and many subclinical cases remain undetected. After the pathogen is inhaled by a host, phagocyted bacilli can persist inside macrophages surviving intracellularly. Hosts develop granulomatous lesions in the lungs or lymph nodes, limiting infection. However, bacilli become persister cells. Immunological diagnosis of TB is performed basically by routine tuberculin skin test (TST), and in some cases, by ancillary interferon-gamma release assay (IGRA). The concept of human latent TB infection (LTBI) by M. tuberculosis is recognized in cohorts without symptoms by routine clinical diagnostic tests, and nowadays IGRA tests are used to confirm LTBI with either active or latent specific antigens of M. tuberculosis. On the other hand, dormant infection in cattle by M. bovis has not been described by TST or IGRA testing as complications occur by cross-reactive immune responses to homolog antigens of environmental mycobacteria or a false-negative test by anergic states of a wained bovine immunity, evidencing the need for deciphering more specific biomarkers by new-generation platforms of analysis for detection of M. bovis dormant infection. The study and description of bovine latent TB infection (boLTBI) would permit the recognition of hidden animal infection with an increase in the sensitivity of routine tests for an accurate estimation of infected dairy cattle. Evidence of immunological and experimental analysis of LTBI should be taken into account to improve the study and the description of the still neglected boLTBI.

Development and Validation of a Tool for the Prediction of Vancomycin-Resistant Enterococci Colonization Persistence-the PREVENT Score.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with increasing prevalence worldwide. Extensive hygiene measures have been established to prevent infection transmission in hospitals. Here, we developed a predictive score system (the predictive vancomycin-resistant enterococci [PREVENT] score) to identify the clearance or persistence in patients with a history of VRE carrier status at readmission. Over a cumulative 3-year period, patients with a positive VRE carrier status were included. The study population was recruited in two successive time periods and separated into training data for predictive score development and validation data for evaluation of the predictive power. The risk factors for persistent VRE colonization were analyzed in a univariable analysis before development of a logistic regression model based on the potential risk factors. The score points were determined proportionally to the beta coefficients of the logistic regression model. The data from 448 (79%) patients were used as the training data, and those from 119 (21%) as the validation data. Multivariable analysis revealed the following variables as independent risk factors: age of ≥60 years, hemato-oncological disease, cumulative antibiotic treatment for >4 weeks, and a VRE infection. The resulting logistic regression model exhibited an acceptable area under the curve (AUC) of 0.81 (95% confidence interval [CI], 0.72 to 0.91). The predictive score system had a sensitivity of 82% (95% CI, 65 to 93%) and a specificity of 77% (95% CI, 66 to 85%). The developed predictive score system is a useful tool to assess the VRE carrier status of patients with a history of VRE colonization. On the basis of this risk assessment, more focused and cost-effective infection control measures can be implemented. IMPORTANCE Given the increasing relevance of VRE as nosocomial pathogens worldwide, infection prevention and control measures, including patient isolation and contact precautions, are indispensable to avoid their spread in the hospital setting. In this study, we developed and validated the PREVENT score, a tool for rapid risk assessment of VRE persistence in patients with a history of previous VRE colonization. The score is designed to be easily performed, employing clinical information available in a regular admission setting and immediately providing information to inform the decision of whether to adopt patient isolation and contact precautions during the hospital stay. After validation, the score was shown to accurately identify patients with persistent VRE colonization upon admission, representing a suitable option as (i) a complementary method yielding preliminary results significantly more quickly than culture-based VRE detection techniques and (ii) an alternative strategy for VRE detection in settings in which microbiological VRE screening is not routinely performed due to limited resources.

The Dynamic Transition of Persistence toward the Viable but Nonculturable State during Stationary Phase Is Driven by Protein Aggregation.

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage, while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation, even at endogenous levels, and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically controlled, gradual development of persisters and VBNC cells through protein aggregation. IMPORTANCE While persistence and the viable but nonculturable (VBNC) state are currently investigated in isolation, our results strongly indicate that these phenotypes represent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and further development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation. Our results thereby strongly hint at a causal relationship between both. Because many conditions known to trigger persistence are also known to influence aggregation, it is tempting to speculate that a variety of different persister pathways converge at the level of protein aggregation. If so, aggregation could emerge as a general principle that underlies the development of persistence which could be exploited for the design of antipersister therapies.

Bacteriophage Therapy for Difficult-to-Treat Infections: The Implementation of a Multidisciplinary Phage Task Force (The PHAGEFORCE Study Protocol).

In times where only a few novel antibiotics are to be expected, antimicrobial resistance remains an expanding global health threat. In case of chronic infections caused by therapy-resistant pathogens, physicians have limited therapeutic options, which are often associated with detrimental consequences for the patient. This has resulted in a renewed interest in alternative strategies, such as bacteriophage (phage) therapy. However, there are still important hurdles that currently impede the more widespread implementation of phage therapy in clinical practice. First, the limited number of good-quality case series and clinical trials have failed to show the optimal application protocol in terms of route of administration, frequency of administration, treatment duration and phage titer. Second, there is limited information on the systemic effects of phage therapy. Finally, in the past, phage therapy has been applied intuitively in terms of the selection of phages and their combination as parts of phage cocktails. This has led to an enormous heterogeneity in previously published studies, resulting in a lack of reliable safety and efficacy data for phage therapy. We hereby present a study protocol that addresses these scientific hurdles using a multidisciplinary approach, bringing together the experience of clinical, pharmaceutical and molecular microbiology experts.

The road to success of coagulase-negative staphylococci: clinical significance of small colony variants and their pathogenic role in persistent infections.

Bacterial small colony variants represent an important aspect of bacterial variability. They are naturally occurring microbial subpopulations with distinctive phenotypic and pathogenic traits, reported for many clinically important bacteria. In clinical terms, SCVs tend to be associated with persistence in host cells and tissues and are less susceptible to antibiotics than their wild-type (WT) counterparts. The increased tendency of SCVs to reside intracellularly where they are protected against the host immune responses and antimicrobial drugs is one of the crucial aspects linking SCVs to recurrent or chronic infections, which are difficult to treat. An important aspect of the SCV ability to persist in the host is the quiescent metabolic state, reduced immune response and expression a changed pattern of virulence factors, including a reduced expression of exotoxins and an increased expression of adhesins facilitating host cell uptake. The purpose of this review is to describe in greater detail the currently available data regarding CoNS SCV and, in particular, their clinical significance and possible mechanisms by which SCVs contribute to the pathogenesis of the chronic infections. It should be emphasized that in spite of an increasing clinical significance of this group of staphylococci, the number of studies unraveling the mechanisms of CoNS SCVs formation and their impact on the course of the infectious process is still scarce, lagging behind the studies on S. aureus SCVs.

Genomic characterization of Achromobacter species isolates from chronic and occasional lung infection in cystic fibrosis patients.

Achromobacter species are increasingly being detected in cystic fibrosis (CF) patients, where they can establish chronic infections by adapting to the lower airway environment. To better understand the mechanisms contributing to a successful colonization by Achromobacter species, we sequenced the whole genome of 54 isolates from 26 patients with occasional and early/late chronic lung infection. We performed a phylogenetic analysis and compared virulence and resistance genes, genetic variants and mutations, and hypermutability mechanisms between chronic and occasional isolates. We identified five Achromobacter species as well as two non-affiliated genogroups (NGs). Among them were the frequently isolated Achromobacter xylosoxidans and four other species whose clinical importance is not yet clear: Achromobacter insuavis, Achromobacter dolens, Achromobacter insolitus and Achromobacter aegrifaciens. While A. insuavis and A. dolens were isolated only from chronically infected patients and A. aegrifaciens only from occasionally infected patients, the other species were found in both groups. Most of the occasional isolates lacked functional genes involved in invasiveness, chemotaxis, type 3 secretion system and anaerobic growth, whereas the great majority (>60%) of chronic isolates had these genomic features. Interestingly, almost all (n=22/23) late chronic isolates lacked functional genes involved in lipopolysaccharide production. Regarding antibiotic resistance, we observed a species-specific distribution of blaOXA genes, confirming what has been reported in the literature and additionally identifying blaOXA-2 in some A. insolitus isolates and observing no blaOXA genes in A. aegrifaciens or NGs. No significant difference in resistance genes was found between chronic and occasional isolates. The results of the mutator genes analysis showed that no occasional isolate had hypermutator characteristics, while 60% of early chronic (<1 year from first colonization) and 78% of late chronic (>1 year from first colonization) isolates were classified as hypermutators. Although all A. dolens, A. insuavis and NG isolates presented two different mutS genes, these seem to have a complementary rather than compensatory function. In conclusion, our results show that Achromobacter species can exhibit different adaptive mechanisms and some of these mechanisms might be more useful than others in establishing a chronic infection in CF patients, highlighting their importance for the clinical setting and the need for further studies on the less clinically characterized Achromobacter species.

Helicobacter pylori Biofilm and New Strategies to Combat it.

Helicobacter pylori, the most frequent pathogen worldwide that colonizes around 50% of the world's population, causes important diseases such as gastric adenocarcinoma, chronic gastritis, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In recent years, various studies have reported that H. pylori biofilm may be one of the critical barriers to the eradication of this bacterial infection. Biofilms inhibit the penetration of antibiotics, increase the expression of efflux pumps and mutations, multiple therapeutic failures, and chronic infections. Nanoparticles and natural products can demolish H. pylori biofilm by destroying the outer layers and inhibiting the initial binding of bacteria. Also, the use of combination therapies destroying extracellular polymeric substances decreases coccoid forms of bacteria and degrading polysaccharides in the outer matrix that lead to an increase in the permeability and performance of antibiotics. Different probiotics, antimicrobial peptides, chemical substances, and polysaccharides by inhibiting adhesion and colonization of H. pylori can prevent biofilm formation by this bacterium. Of note, many of the above are applicable to acidic pH and can be used to treat gastritis. Therefore, H. pylori biofilm may be one of the major causes of failure to eradication of infections caused by this bacterium, and antibiotics are not capable of destroying the biofilm. Thus, it is necessary to use new strategies to prevent recurrent and chronic infections by inhibiting biofilm formation.

Microbiome and Cervical Cancer.

Persistent infection with some types of mucosal human papillomavirus (HPV) is the etiological factor for the development of cervical cancer and its precursor lesions. Besides, several cofactors are known to play a role in cervical disease onset and progression either by favoring or by preventing HPV infection and persistence. The microbiome of a healthy female genital tract is characterized by the presence of 1 or few varieties of lactobacilli. However, high-throughput studies addressing the bacterial diversity and abundance in the female genital tract have shown that several factors, including hormonal levels, hygiene habits, and sexually transmitted diseases may disrupt the natural balance, favoring the outgrowth of some groups of bacteria, which in turn may favor some pathological states. Recently, the vaginal microbiome has emerged as a new variable that could greatly influence the natural history of HPV infections and their clinical impact. In this context, changes in the vaginal microbiome have been detected in women infected with HPV and women with HPV-associated lesions and cancer. However, the role of specific bacteria groups in the development/progression or prevention/regression of HPV-associated pathologies is not well understood. In this review we summarize the current knowledge concerning changes in vaginal microbiome and cervical disease. We discuss the potential functional interplay between specific bacterial groups and HPV infection outcomes.