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Pseudomonas aeruginosa is a major, but opportunistic, respiratory pathogen, which rarely infects healthy individuals, mainly due to the barrier effect of the human airway epithelium (HAE). This review explores the interaction of P . aeruginosa with HAE and the progression of the infection. The basolateral part of the epithelium, which includes the basolateral membrane of the epithelial cells and the basement membrane, is inaccessible in normal tight epithelia with intact junctions. We highlight how P . aeruginosa exploits weaknesses in the HAE barrier to gain access to the basolateral part of the epithelium. This access is crucial to initiate respiratory infection and is mainly observed in the injured epithelium, in repairing or chronically remodeled epithelium, and during extrusion of senescent cells or cell multiplication during normal epithelium renewal. The subsequent adhesion of the bacteria and cytotoxic action of virulence factors, including the toxins delivered by the type 3 secretion system (T3SS), lead to retractions and cell death. Eventually, P . aeruginosa progressively reaches the basement membrane and propagates radially through the basal part of the epithelium to disseminate using twitching and flagellar motility.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa . To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the V H and/or V L -chain of a monoclonal antibody, VSX, that targets the core of P . aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P . aeruginosa strains, and protects mice from P . aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H 2 O 2 ) and hypothiocyanite (OSCN - ), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H 2 O 2 exposure that does not recapitulate the complex H 2 O 2 /LPO/SCN - system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H 2 O 2 /OSCN - concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H 2 O 2 /OSCN - were steadily produced from 7h and up to 25h, but OSCN - was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H 2 O 2 of roughly 50% for 10 5 and 10 7 colony-forming unit (CFU)/mL inocula, while 10 2 and 10 4 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P . aeruginosa , S . aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.

An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism’s overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa , treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD ⁺ strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition characterized by chronic respiratory symptoms due to abnormalities of the airways and/or alveoli that cause airflow obstruction. It is a leading cause of death worldwide. While alterations in airway bacterial community have been linked to exacerbation frequency, the underlying mechanisms remain unclear. We aimed to characterize associations between airway bacterial community structure and exacerbation history in stable COPD patients and to identify candidate microbial markers that could assist in risk assessment for the clinical management of COPD patients. Sixty-two stable COPD patients were enrolled and categorized into two groups based on their exacerbation history: low risk (LR) and high risk (HR) of exacerbation. Sputum samples were collected and analyzed using both bacterial extended culture and 16S rRNA gene sequencing. The combination of these approaches provided complementary insights, enabling a more comprehensive characterization of the bacterial community. To our knowledge, this is the first study to combine these two approaches in this context and to evaluate their relative performance in detecting microbiological markers associated with exacerbation risk. Microbial composition analysis revealed a loss of α-diversity in HR patients based on extended culture data, a finding not corroborated by sequencing. This discrepancy suggests that the observed impoverishment of diversity may primarily affect the viable fraction of the airway microbial community. The HR group also exhibited increased relative abundances of Pseudomonadota and Bacteroidota, alongside a marked decrease in relative abundances of Lactobacillus and Streptococcus . Notably, significant reductions in the proportion of positive samples were observed at the species level for Streptococcus salivarius and Streptococcus mutans . A comparison of the two methods underlined that 16S rRNA gene sequencing identified five additional phyla and 84 genera not detected by culture, notably strict anaerobes. However, extended culture demonstrated robust sensitivity in detecting Enterobacterales and the pathogenic Moraxella and Pseudomonas . This study revealed microbiological features linked to exacerbation history in stable COPD patients, highlighting the need for future functional and longitudinal research to validate these airway bacterial community signatures and develop targeted preventive strategies.

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli , sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD -plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD . Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide ( FAD )-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase ( FNR )-type transcription factor, related to an O 2 -responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli . Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies. FIVE SAND FLY SPECIES: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates. Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.

Background Cough and sputum are major symptoms in cystic fibrosis (CF) that contribute to the impairment of quality of life. Methods This prospective single centre cross-sectional pilot study aimed to evaluate the results of a self-administered questionnaire assessing cough and sputum symptoms (2 domains), and their impact (2 domains) on daily activities in the previous week, named the Cough and Sputum Assessment Questionnaire (CASA-Q) in CF adult patients at stable state, and to analyse associations with clinical, functional, microbiological, radiological data, and two quality of life scales: the Cystic Fibrosis Questionnaire Revised (CFQ-R) and the Saint George Respiratory Questionnaire (SGRQ). Results Forty-eight patients were included in this analysis (69% men; median age of 27.8  ±  8.1 years; median body mass index of 21.8  +  3.3 kg/m²; mean FEV 1 of 64  ±  30% of the predicted value). The mean values of the CASA-Q domains were 58  ±  23 for cough symptoms, 77  ±  24 for cough impact, 62  ±  25 for sputum symptoms and 84  ±  21 for sputum impact. Impairment in CASA-Q cough and sputum domains was associated with dyspnea mMRC scale (p < 0.005 for all 4 domains of CASA-Q) and exacerbations in the previous year (p < 0.05 for CASA-Q symptoms domains). We also found correlations between all domains of the CASA-Q and quality of life questionnaires including SGRQ (p < 0.001) and to a lesser extend CFQ-R. We identified a clinical phenotype (female gender, ΔF508 heterozygous mutation, dyspnea mMRC scale) associated with an impairment of CASA-Q score and quality of life using a 2-step cluster analysis. Conclusions CASA-Q allows the assessment of cough and sputum in CF adult patients and is associated with quality of life impairment. This simple easy-to-use tool could be used in routine clinical practice and in clinical studies to assess cough and sputum in CF patients. Trial Registration The study was registered on ClinicalTrials.gov (NCT02924818, first posted on 5th October 2016).

Background Bronchiectasis is a chronic airway disease characterized by permanent and irreversible abnormal dilatation of bronchi. Several studies have reported the development of bronchiectasis after renal transplantation (RT), but no prospective study specifically assessed bronchiectasis in this population. This study aimed to compare features of patients with bronchiectasis associated with RT to those with idiopathic bronchiectasis. Methods Nineteen patients with bronchiectasis associated with RT (RT-B group) and 23 patients with idiopathic bronchiectasis (IB group) were prospectively included in this monocentric cross-sectional study. All patients underwent clinical, functional, laboratory, and CT scan assessments. Sputum was collected from 25 patients ( n  = 11 with RT-B and n  = 14 with IB) and airway microbiota was analyzed using an extended microbiological culture. Results Dyspnea (≥ 2 on mMRC scale), number of exacerbations, pulmonary function tests, total bronchiectasis score, severity and prognosis scores (FACED and E-FACED), and quality of life scores (SGRQ and MOS SF-36) were similar in the RT-B and IB groups. By contrast, chronic cough was less frequent in the RT-B group than in the IB group (68% vs. 96%, p  = 0.03). The prevalence and diversity of the airway microbiota in sputum were similar in the two groups. Conclusion Clinical, functional, thoracic CT scan, and microbiological characteristics of bronchiectasis are overall similar in patients with IB and RT-B. These results highlight that in RT patients, chronic respiratory symptoms and/or airway infections should lead to consider the diagnosis of bronchiectasis. Further studies are required to better characterize the pathophysiology of RT-B including airway microbiota, its incidence, and impact on therapeutic management.

Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) has become a staple in clinical microbiology laboratories. Protein‐profiling of bacteria using this technique has accelerated the identification of pathogens in diagnostic workflows. Recently, lipid profiling has emerged as a way to complement bacterial identification where protein‐based methods fail to provide accurate results. This study aimed to address the challenge of rapid discrimination between Escherichia coli and Shigella spp. using MALDI‐TOF MS in the negative ion mode for lipid profiling coupled with machine learning. Both E. coli and Shigella species are closely related; they share high sequence homology, reported for 16S rRNA gene sequence similarities between E. coli and Shigella spp. exceeding 99%, and a similar protein expression pattern but are epidemiologically distinct. A bacterial collection of 45 E. coli, 48 Shigella flexneri, and 62 Shigella sonnei clinical isolates were submitted to lipid profiling in negative ion mode using the MALDI Biotyper Sirius® system after treatment with mild‐acid hydrolysis (acetic acid 1% v/v for 15 min at 98°C). Spectra were then analyzed using our in‐house machine learning algorithm and top‐ranked features used for the discrimination of the bacterial species. Here, as a proof‐of‐concept, we showed that lipid profiling might have the potential to differentiate E. coli from Shigella species using the analysis of the top five ranked features obtained by MALDI‐TOF MS in the negative ion mode of the MALDI Biotyper Sirius® system. Based on this new approach, MALDI‐TOF MS analysis of lipids might help pave the way toward these goals. Workflows for the identification of shigellosis in a clinical microbiology laboratory. The routine workflow is represented by a black arrow while the lipid profiling identification workflow is represented by green arrows. Combined with a machine learning algorithm, lipid profiling by routine MALDI in the negative ion mode might have the potential to differentiate Escherichia coli from Shigella species.