Chronic Lung Infections In Patients Research Articles

The Impact of CFTR Modulating Therapy on Chronic Lung Infection in Patients with Cystic Fibrosis

Cystic fibrosis is the most common lethal genetic disease in the white population, affecting approximately 80 000 people worldwide. It is an autosomal recessive, monogenic, and multisystemic disease, with over 2000 mutations described in the CFTR protein gene. The dysfunction of this protein leads to a decrease in the secretion of chlorine and bicarbonate, sodium hyperabsorption, and consequent water absorption, resulting in the thickening of secretions and accumulation of pathogens. These changes culminate in inflammation, chronic pulmonary infection, and recurrent exacerbations, with lung disease being the main cause of morbidity and mortality. In the early stages of the disease, Staphylococcus aureus is generally the agent responsible for chronic infection. Over time, Pseudomonas aeruginosa becomes more prevalent, being the most frequent bacteria in adults. However, in up to 70% of patients, colonization is polymicrobial, with frequent isolation of S. aureus and P. aeruginosa, associated with Haemophilus influenzae or Streptococcus pneumoniae, as well as isolation of other bacterial agents, viruses, or fungi. In recent years, drugs modulating CFTR have been developed which have shown a positive effect on lung function, body mass index, exacerbation rate, chlorine concentration, and quality of life. Currently, four drugs are approved that act by improving the function or increasing the amount of protein produced and consequently the ion transport. [...].

P148 Mixed bacterial-viral lung infections in patients with cystic fibrosis (CF) during an exacerbation

Chronic lung infection in patients with cystic fibrosis in more than 70% of cases is mixed. About 40% of CF patients during exacerbations of a chronic lung infection require hospital treatment. CF, like other congenital pathologies of the respiratory system, is a risk factor for a more unfavourable course of viral infections of the respiratory tract.

Antibacterial Activity of a Natural Clay Mineral against Burkholderia cepacia Complex and Other Bacterial Pathogens Isolated from People with Cystic Fibrosis.

There is an impending crisis in healthcare brought about by a new era of untreatable infections caused by bacteria resistant to all available antibiotics. Thus, there is an urgent need to identify novel antimicrobial agents to counter the continuing threat posed by formerly treatable infections. We previously reported that a natural mineral clay known as Kisameet clay (KC) is a potent inhibitor of the organisms responsible for acute infections. Chronic bacterial infections present another major challenge to treatment by antimicrobials, due to their prolonged nature, which results in repeated exposure to antibiotics and a constant selection for antimicrobial resistance. A prime example is bacteria belonging to the Burkholderia cepacia complex (Bcc), which particularly causes some of the most serious chronic lung infections in patients with cystic fibrosis (CF) associated with unpredictable clinical outcomes, poor prognosis, and high mortality rates. Eradication of these organisms from CF patients with limited effective antimicrobial options is a major challenge. Novel therapeutic approaches are urgently required. Here, we report the in vitro antibacterial activity of KC aqueous suspensions (1-10% w/v) and its aqueous extract (L100) against a collection of extensively and multi-drug resistant clinical isolates of Bcc, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia isolated from patients with CF. These findings present a potential novel therapy for further investigation in the clinic.

Achromobacter spp. Adaptation in Cystic Fibrosis Infection and Candidate Biomarkers of Antimicrobial Resistance

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

P128 Epidemiological significance of Achromobacter spp. chronic lung infection in patients with cystic fibrosis

P128 Epidemiological significance of Achromobacter spp. chronic lung infection in patients with cystic fibrosis

A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology.

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

19 Nebulized Tobramycin is Associated with Decreased Pneumonia After Inhalation Injury

Abstract Introduction Management of inhalation injury is largely supportive and consists primarily of mechanical ventilation, bronchodilators, muscarinic receptor antagonists, and inhaled mucolytics and anticoagulants. Patients with inhalation injury are at high risk for pneumonia. Nebulized tobramycin is well supported in the use of chronic lung infections in patients with cystic fibrosis (CF). We hypothesize that after inhalation injury patients empirically treated with nebulized tobramycin (NT) have decreased incidence of pneumonia. Methods A protocol for a standardized 7-day course of nebulized treatments with bronchodilators, inhaled mucolytics combined with inhaled anticoagulants and Nebulized tobramycin was developed. Starting in May 2013, all patients that had a clinical diagnosis of inhalation documented by the attending physician were started on the 7-day treatment course. Patients with inhalation injury from 2009–2019 were retrospectively reviewed for treatment of inhalation injury. Univariate analysis and multiple logistic regression were performed using Stata. Results Of 90 patients with inhalation injury, median age was 52 (IQR:31–60) with 27% (n=23) women and median TBSA of 10%(IQR:0.7–35%). The median length of stay was 21 days (IQR:6–47). Median ventilator days were lower in patients treated with NT (8.5, IQR:4–21) compared to patients that did not receive NT (10, IQR:3–23) but was not significant (p=0.85). However, of those that received NT, the presence of pneumonia was significantly lower compared to patients not treated with NT (p=0.032). (Table 1) Patients who were not treated with NT developed pneumonia earlier (day 5, IQR:3–7) than patients treated with NT (day 10, IQR 6–16, p=0.02) After adjusting for grade of inhalation injury, patients who did not receive NT were 2.7 times as likely to get pneumonia compared to patients who received NT (p=0.037). Conclusions NT is a prophylactic strategy for pneumonia in patients with CF, well documented to be safe. It allows for concentrated antibacterial coverage localized to the area of infection without significant systemic absorption. After seeing ~60% incidence of pneumonia in inhalation patients, we developed a strategy that avoided systemic prophylactic treatment. Initial data shows empiric administration of NT in patients with inhalation injury may abrogate the development pneumonia. Additional research and clinical trials are needed to better understand the role of inhaled tobramycin in the management of inhalation injury.

Pseudomonas Aeruginosa Induced Cell Death in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for the cause of acute lung injury and acute respiratory distress syndrome. P. aeruginosa isthe leading species isolated from patients with nosocomial infection and is detected in almost all the patients with long term ventilation in critical care units. P. aeruginosa infection is also the leading cause of deleterious chronic lung infections in patients suffering from cystic fibrosis as well as the major reason for morbidity in people with chronic obstructive pulmonary disease. P. aeruginosa infections are linked to diseases with high mortality rates and are challenging for treatment, for which no effective remedies have been developed. Massive lung epithelial cell death is a hallmark of severe acute lung injury and acute respiratory distress syndrome caused by P. aeruginosa infection. Lung epithelial cell death poses serious challenges to air barrier and structural integrity that may lead to edema, cytokine secretion, inflammatory infiltration, and hypoxia. Here we review different types of cell death caused by P. aeruginosa serving as a starting point for the diseases it is responsible for causing. We also review the different mechanisms of cell death and potential therapeutics in countering the serious challenges presented by this deadly bacterium.

Directions of Epidemiological Surveillance of Chronic Lung Infections Caused by <i>Burkholderia cepacia Complex</i>, <i>Achromobacter spp., Pseudomonas aeruginosa</i> and Methicillin-Resistant <i>Staphylococcus aureus</i> in Patients with Cystic Fibrosis

Relevance. The absence of a system of epidemiological surveillance of chronic lung infection (CLI) in patients with cystic fibrosis (CF) led to the spread of Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex and Achromobacter spp. among patients with CF during hospitalizations, that is proved by numerous cases of infection in patients with CF treated in various Russian clinics. Aims. Determine the directions of epidemiological surveillance (ES) for CLI in patients with CF and preventive measures necessary to counteract the cross-infections among patients with CF and the spread of the dominant pathogens of CLI in the hospital. Materials and methods. A complex of epidemiological, microbiological and molecular genetic methods was used. Results and discussion . Based on the results of studies of the epidemiological and microbiological features of infections caused by the above-mentioned pathogens, the directions of ES and preventive measures for CF were determined. The tasks of the information-analytical unit, including the monitoring of morbidity and microbiological monitoring, are substantiated. It has been established that in the framework of microbiological monitoring, it is necessary to conduct bacteriological examination of biomaterial from the respiratory tract of patients with CF not less than 1 time per quarter, as well as at each outpatient visit and at hospitalization. The necessity of studying the phenotypic properties of pathogens contributing to long-term persistence in the patient's body of the CF, the definition and monitoring of the antibiotic sensitivity of microorganisms isolated from the patient over time, and the study of hypermutability was shown. One of the main tasks of monitoring is molecular genetic analysis of pathogens by PCR for the purpose of intraspecies identification, typing of pathogens and detection of epidemic markers and clones (including international ones), identification of genetic determinants of antibiotic resistance. Conclusion. The introduction of ES for chronic lung infection will improve the quality of etiological diagnosis, will contribute early identification of the sources of infections and prevent the spread of CLI pathogens among patients with CF in hospitals and in non-hospital conditions, as well as optimize the tactics of CLI antimicrobial therapy. The main directions of preventing the spread of CLI pathogens should be the separation of patients with various infections during outpatient examination and isolation during hospitalization.

Burkholderia multivorans Exhibits Antibiotic Collateral Sensitivity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters.

Efficacy of Aerosolized Rifaximin versus Tobramycin for Treatment of Pseudomonas aeruginosa Pneumonia in Mice.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronic lung infections in patients with cystic fibrosis (CF). The current preferred treatment for CF lung infections includes inhaled tobramycin (TOB); however, studies suggest TOB cannot effectively inhibit biofilm formation. Using an NIH small compounds drug library approved for safe use in humans, we identified rifaximin (RFX), a semisynthetic, rifamycin family, nonsystemic antibiotic that inhibits alginate production and growth in P. aeruginosa Inhibition of alginate production was further analyzed using the uronic acid carbazole assay and a promoter reporter assay that measures the transcription of the alginate biosynthetic operon. Compared to TOB, RFX significantly reduced alginate production in laboratory and CF sputum isolates of P. aeruginosa In addition, RFX showed a narrow range of MICs when measured with multidrug-resistant bacterial species of clinical relevance, synergistic activities with TOB or amikacin against clinical isolates, as well as reduction toward in vitro preformed biofilms. In C57BL/6 mice, penetration of nebulized TOB into the lungs was shown at a higher level than that of RFX. Further, in vivo assessment using a DBA/2 mouse lung infection model found increased survival rates with a single-dose treatment of nebulized RFX and decreased P. aeruginosa PAO1 bioburden with a multiple-dose treatment of RFX plus TOB. In addition, mice treated with a single exposure to dimethyl sulfoxide (DMSO), a solvent that dissolves RFX, showed no apparent toxicity. In summary, RFX may be used to supplement TOB inhalation therapy to increase efficacy against P. aeruginosa biofilm infections.

Epidemiological significance of genome variations in Pseudomonas aeruginosa causing chronic lung infection in patients with cystic fibrosis

Objective. To present the data on the main mechanism of molecular variation in P. aeruginosa causing chronic lung infection in patients with cystic fibrosis. Materials and Methods. A total of 1800 throat swabs and sputum samples from cystic fibrosis patients were included in the study over the 10-year period. P. aeruginosa isolates were primarily identified by the biochemical method using the API 20NE test strips (bioMerieux, France). Antimicrobial susceptibility testing was performed by disc diffusion method. Genotyping was conducted by RAPD-PCR and MLST. Whole genome sequencing of three typical P. aeruginosa isolates was performed on an Ion PGM Torrent platform with Ion Sequencing Kit and 316v1 chips (Life Technologies Thermo Fisher, US) according to the manufacturer’s protocol. The RAST web application was used for initial annotation. Results. There were three main variants of the pathogen variability found: population heterogeneity, pathogen microevolution, and replacement by another genotype of the same species. The variation of the pathogen’s genome is due to the acquisition of mobile genetic elements (plasmids), mutations in the chromosomal genes responsible for antibiotic resistance, bacterial viability and survival during persistence in a host, and changes in the prophage regions of the pathogen. Conclusions. Epidemiological significance of the molecular mechanisms of pathogen variation is primarily due to the ability of strains to form epidemiologically significant clone. This requires control measures aimed to limit emergence and distribution of such clones to be developed.

Peptide dendrimers as "lead compounds" for the treatment of chronic lung infections by Pseudomonas aeruginosa in cystic fibrosis patients: in vitro and in vivo studies.

AimIn the present work, the potential of the D-enantiomeric dendrimers dG3KL and dTNS18 was evaluated in relation to tobramycin (Tob), for the development of novel antibacterials to treat Pseudomonas aeruginosa chronic lung infections in patients with cystic fibrosis.ResultsThe activity of dendrimers against planktonic P. aeruginosa cells was less than Tob against three of the four strains tested (median minimum inhibitory concentration [MIC] 8 vs 1 µg/mL, respectively), but 32-fold higher against the PaPh32 strain isolated at posttransplantation stage. Results from comparative minimum bactericidal concentration/MIC evaluation and time–kill assay suggested a bactericidal mechanism for all test agents. Subinhibitory concentrations of both dendrimers and Tob significantly affected biofilm formation by all strains in a dose-dependent manner, although the PaPh26 strain, isolated during the chronic stage of infection, was particularly susceptible to dendrimers. The activity of dendrimers against preformed P. aeruginosa biofilm was generally comparable to Tob, considering both dispersion and viability of biofilm. Particularly, exposure to the test agent at 10 × MIC caused significant biofilm death (>90%, even to eradication), though with strain-specific differences. Single administration of dendrimers or Tob at 10 × MIC was not toxic in Galleria mellonella wax-moth larvae over 96 hours. However, contrarily to Tob, dendrimers were not protective against systemic infection caused by P. aeruginosa in G. mellonella. Kinetics of P. aeruginosa growth in hemolymph showed that bacterial load increased over time in the presence of dendrimers.ConclusionOverall, our findings indicated that dG3KL and dTNS18 peptide dendrimers show in vitro activity comparable to Tob against both P. aeruginosa planktonic and biofilm cells at concentrations not toxic in vivo. Further studies are warranted to explore different dosages and to increase the bioavailability of the peptides to solve the lack of protective effect observed in G. mellonella larvae.

Polymorphonuclear Leukocytes or Hydrogen Peroxide Enhance Biofilm Development of MucoidPseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium involved in many human infections, including pneumonia, diabetic foot ulcers, and ventilator-associated pneumonia. P. aeruginosa cells usually undergo mucoid conversion during chronic lung infection in patients with cystic fibrosis (CF) and resist destruction by polymorphonuclear leukocytes (PMNs), which release free oxygen radicals (ROS), such as H2O2. PMNs are the main leucocytes in the CF sputum of patients who are infected with P. aeruginosa, which usually forms biofilms. Here, we report that PMNs or H2O2 can promote biofilm formation by mucoid P. aeruginosa FRD1 with the use of the hanging-peg method. The mucoid strain infecting CF patients overproduces alginate. In this study, PMNs and H2O2 promoted alginate production, and biofilms treated with PMNs or H2O2 exhibited higher expression of alginate genes. Additionally, PMNs increased the activity of GDP-mannose dehydrogenase, which is the key enzyme in alginate biosynthesis. Our results demonstrate that PMNs or H2O2 can enhance mucoid P. aeruginosa biofilms.

Epidemiological Features of Chronic Lung Infection in Patients with Cystic Fibrosis

Relevance. Life expectancy of cystic fibrosis patients mostly depends on the degree of respiratory system damage caused by opportunistic microorganisms, which is due to the fact that 90-95% of deaths of cystic fibrosis patients are caused by lung infections. Goal. To define epidemiologic characteristics of chronic lung infection caused by the most common agents (S. aureus, P. aeruginosa, B. cepacia-like bacteria (Bcc) and Achromobacter spp.) using a novel chronic lung infection in cystic fibrosis patients microbiological diagnosis algorithm. Materials and methods. Over a period of 7 years (2008-2016) 300 children with cystic fibrosis living in Moscow, Moscow region and several other regions of Russian Federation have been checked-up. 260 sputum samples from 100 adult patients, who were under care at the Pulmonology Research Institute, were studied. Sputum samples from children were taken before and after antibiotic therapy with intervals of 15-45 days and over 6 months. 30 of the children were also subjected to a microbiologic monitoring of the state of chronic infection in the period between 4 and 15 months. Sputum sample from adult patients were also taken before and after antibiotic therapy with intervals of 0, 15-45 days and over 6 months. Results. P. aeruginosa, S. aureus, H. influenzae and Burkholderia cepacia-like bacteria were confirmed to be the most common agents of lung infection in cystic fibrosis patients. Children with cystic fibrosis over the years develop foci of chronic lung infection, mainly caused by P. aeruginosa and S. aureus. Conclusions. Chronic lung infection can be caused by community-acquired or nosocomial S. aureus и P. aeruginosa. Chronic lung infection is a complex, dynamically changing disease which requires constant monitoring and is mainly caused by S. aureus, P. aeruginosa, Bcc bacteria and Achromobacter spp. As populations of the agents can be diverse, it is necessary to study all colonies with differing phenotypes (mucoid and non-mucoid variants, small colony variants, variants with different pigments) and to take samples of several colonies when testing antibiotic resistance. Bcc and Achromobacter spp. cannot be eradicated with antibiotics, thus the only effective measure against these bacteria can only be vaccination which requires developing a vaccine.

An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.

In Vitro Analysis of Pseudomonas aeruginosa Virulence Using Conditions That Mimic the Environment at Specific Infection Sites.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic lung infection in patients with cystic fibrosis (CF) and acute systemic infections in severely burned patients and immunocompromised patients including cancer patients undergoing chemotherapy and HIV infected individuals. In response to the environmental conditions at specific infection sites, P. aeruginosa expresses certain sets of cell-associated and extracellular virulence factors that produce tissue damage. Analyzing the mechanisms that govern the production of these virulence factors in vitro requires media that closely mimic the environmental conditions within the infection sites. In this chapter, we review studies based on media that closely resemble three in vivo conditions, the thick mucus accumulated within the lung alveoli of CF patients, the serum-rich wound bed and the bloodstream. Media resembling the CF alveolar mucus include standard laboratory media supplemented with sputum obtained from CF patients as well as prepared synthetic mucus media formulated to contain the individual components of CF sputum. Media supplemented with serum or individual serum components have served as surrogates for the soluble host components of wound infections, while whole blood has been used to investigate the adaptation of pathogens to the bloodstream. Studies using these media have provided valuable information regarding P. aeruginosa gene expression in different host environments as varying sets of genes were differentially regulated during growth in each medium. The unique effects observed indicate the essential role of these in vitro media that closely mimic the in vivo conditions in providing accurate information regarding the pathogenesis of P. aeruginosa infections.

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression.

Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity.

Fosfomycin as an alternative therapeutic option for treatment of infections caused by multi-resistant Gram-negative bacteria

The problem of significantly reduced drug use concerns particularly the infections caused by multi-resistant pathogens, especially Gram-negative bacteria. In this regard, interest is increasing in the known for nearly 50 years, but now frequently forgotten antibiotic – fosfomycin. Fosfomycin possesses high effectiveness for multidrug-resistant bacteria, comprising of extended-spectrum β-lactamases (ESBL), Klebsiella pneumoniae carbapenemases (KPC); commonly, the pandrug-resistant (PDR) and the extensively drug-resistant (XDR) strains, especially from Enterobacteriaceae family. Because of facilitated distribution into inflamed tissues and a very broad range of in vitro bactericidal activity, fosfomycin may have various applications in the treatment of many kinds of bacterial infections, including acute uncomplicated infections of the urinary bladder or complicated urinary tract infections, urinary tract sepsis, pyelonephritis, cystitis, prostatitis and chronic lung infections in patients with cystic fibrosis, the great majority caused by multi-resistant Gram-negative bacteria, and in the therapy eradicating multidrug-resistant Helicobacter pylori. Fosfomycin may limit the toxicity of other antibiotics and play a protective role in the process of bacterial resistance development during the therapy. Combinations of different antimicrobials enable the use of forgotten antibiotics in commonly occurring infections, although they are still completely incurable.