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Elexacaftor/tezacaftor/ivacaftor (ETI) therapy has revolutionized the treatment of cystic fibrosis (CF) for most affected individuals but the effects of treatment on sinus microbiota are still unknown. Changes to the airway microbiota in CF are associated with disease state and alterations to the bacterial community after ETI initiation may require changes to clinical management regimens. We collected sinus swab samples from the middle meatus in an observational study of 38 adults with CF and chronic rhinosinusitis (CRS) from 2017 to 2021 and captured the initiation of ETI therapy. We performed 16S and custom amplicon sequencing to characterize the sinus microbiota pre- and post-ETI. Real-time quantitative PCR (RT-qPCR) was performed to estimate total bacterial abundance. Sinus samples from people with CF (pwCF) clustered into three community types, dependent on the dominant bacterial organism: a Pseudomonas -dominant, Staphylococcus -dominant, and mixed dominance cluster. Shannon's diversity index was low and not significantly altered post-ETI. Total bacterial load was not significantly lowered post-ETI. Pseudomonas spp. abundance was significantly reduced post-ETI, but eradication was not observed. Staphylococcus spp. became the dominant organism in most individuals post-ETI and we showed the presence of methicillin-resistant Staphylococcus aureus (MRSA) in the sinus both pre- and post-ETI. We also demonstrated that the sinus microbiome is predictive of the presence of Pseudomonas spp., Staphylococcus spp., and Serratia spp. in the sputum. Pseudomonas spp. and Staphylococcus spp., including MRSA, persist in the sinuses of pwCF after ETI therapy, indicating that these pathogens will continue to be important in CF airway disease management in the era of highly effective modulator therapies (HEMT).IMPORTANCEHighly effective modulator therapies (HEMT), such as elexacaftor/tezacaftor/ivacaftor (ETI), for cystic fibrosis (CF) have revolutionized patient care and quality of life for most affected individuals. The effects of these therapies on the microbiota of the airways are still unclear, though work has already been published on changes to microbiota in the sputum. Our study presents evidence for reduced relative abundance of Pseudomonas spp. in the sinuses following ETI therapy. We also show that Staphylococcus spp. becomes the dominant organism in the sinus communities of most individuals in this cohort after ETI therapy. We identified methicillin-resistant Staphylococcus aureus (MRSA) in the sinus microbiota both pre- and post-therapy. These findings demonstrate that pathogen monitoring and treatment will remain a vital part of airway disease management for people with cystic fibrosis (pwCF) in the era of HEMT., Competing Interests: The authors declare no conflict of interest.

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rapsinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Today, more than 90% of people with cystic fibrosis (pwCF) are eligible for the highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy called elexacaftor/tezacaftor/ivacaftor (ETI) and its use is widespread. Given the drastic respiratory symptom improvement experienced by many post-ETI, clinical studies are already underway to reduce the number of respiratory therapies, including antibiotic regimens, that pwCF historically relied on to combat lung disease progression. Early studies suggest that bacterial burden in the lungs is reduced post-ETI, yet it is unknown how chronic Pseudomonas aeruginosa populations are impacted by ETI. We found that pwCF remain infected throughout their upper and lower respiratory tract with their same strain of P. aeruginosa post-ETI, and these strains continue to evolve in response to the newly CFTR-corrected airway. Our work underscores the continued importance of CF airway microbiology in the new era of highly effective CFTR modulator therapy., Importance: The highly effective cystic fibrosis transmembrane conductance regulator modulator therapy Elexakaftor/Tezacaftor/Ivacaftor (ETI) has changed cystic fibrosis (CF) disease for many people with cystic fibrosis. While respiratory symptoms are improved by ETI, we found that people with CF remain infected with Pseudomonas aeruginosa . How these persistent and evolving bacterial populations will impact the clinical manifestations of CF in the coming years remains to be seen, but the role and potentially changing face of infection in CF should not be discounted in the era of highly effective modulator therapy., Competing Interests: The authors declare no conflict of interest.

Background: While the widespread initiation of elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic clinical improvements among persons with cystic fibrosis (pwCF), little is known about how ETI affects the respiratory mucosal inflammatory and physiochemical environment, or how these changes relate to lung function., Methods: We performed a prospective, longitudinal study of adults with CF and chronic rhinosinusitis (CF-CRS) followed at our CF center (n = 18). Endoscopic upper respiratory tract (paranasal sinus) aspirates from multiple visit dates, both pre- and post-ETI initiation, were collected and tested for cytokines, metals, pH, and lactate levels. Generalized estimating equations were used to identify relationships between ETI and upper respiratory tract (URT) biomarker levels, and between URT biomarkers and lung function or clinical sinus parameters., Results: ETI was associated with decreased upper respiratory mucosal cytokines B-cell activating factor (BAFF), IL-12p40, IL-32, IL-8, IL-22 and soluble tumor necrosis factor-1 (sTNFR1), and an increase in a proliferation-inducing ligand (APRIL) and IL-19. ETI was also associated with decreased URT levels of copper, manganese, and zinc. In turn, lower URT levels of BAFF, IL-8, lactate, and potassium were each associated with ~1.5% to 4.3% improved forced expiratory volume in 1 s (FEV 1 ), while higher levels of IFNγ, iron, and selenium were associated with ~2% to 10% higher FEV 1 ., Conclusions: Our observations suggest a dampening of inflammatory signals and restriction in microbial nutrients in the upper respiratory tract with ETI. These findings improve our understanding of how ETI impacts the mucosal environment in the respiratory tract, and may give insight into the improved infectious and inflammatory status and the resulting clinical improvements seen in pwCF., (© 2024 Wiley Periodicals LLC.)

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses., Competing Interests: PET is cofounder of Felix Biotechnology, Inc., a company that seeks to develop phages for human therapy. Yale University has an institutional conflict of interest related to this project (PET & JLK). Yale may receive financial benefit related to the therapy used in this protocol., (Copyright: © 2024 Zamora et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1β [IL-1β]). Increased IL-1β was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1β in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1β. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Background: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences., Results: While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semi-automated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias., Conclusions: The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events., (© 2022. The Author(s).)

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution., Competing Interests: Declaration of interests V.S.C. is a founder of Microbial Genome Sequencing Center, LLC (MiGS) and is a scientific advisor for MiGS., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy., Objectives: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present., Methods: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing., Results: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content., Conclusions: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently used a plasmid-based c-di-GMP reporter (pP cdrA ::gfp ) to describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells during early biofilm formation. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. Here, we describe the protocol for a key experiment to confirm our initial observation of c-di-GMP heterogeneity during surface sensing: the use of flow-assisted cell sorting (FACS) to isolate subpopulations of cells with high and low c-di-GMP reporter activity, followed by quantitative Reverse Transcriptase PCR (qRT-PCR) of genes that are known to be transcriptionally regulated in response to cellular c-di-GMP levels ( pelA, pslA ). This protocol can be adapted by others to isolate subpopulations of high- and low- c-di-GMP P. aeruginosa cells that are genetically identical, but phenotypically distinct for future experiments examining specific mRNA transcripts as we did or, presumably, for additional applications like RNAseq, proteomics, or TNseq. Graphical abstract ., Competing Interests: Competing interestsThe authors have no competing interests to declare., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

The respiratory tract of individuals with cystic fibrosis is host to polymicrobial infections that persist for decades and lead to significant morbidity and mortality. Improving our understanding of CF respiratory infections requires coordinated efforts from researchers in the fields of microbial physiology, genomics, and ecology, as well as epithelial biology and immunology. Here, we have highlighted examples from recent CF microbial pathogenesis literature of how the host nutritional environment, immune response, and microbe-microbe interactions can feedback onto each other, leading to diverse effects on lung disease pathogenesis in CF., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

What are bacteria doing during "reversible attachment," the period of transient surface attachment when they initially engage a surface, besides attaching themselves to the surface? Can an attaching cell help any other cell attach? If so, does it help all cells or employ a more selective strategy to help either nearby cells (spatial neighbors) or its progeny (temporal neighbors)? Using community tracking methods at the single-cell resolution, we suggest answers to these questions based on how reversible attachment progresses during surface sensing for Pseudomonas aeruginosa strains PAO1 and PA14. Although PAO1 and PA14 exhibit similar trends of surface cell population increase, they show unanticipated differences when cells are considered at the lineage level and interpreted using the quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two surface colonization strategies. PAO1 lineages commit quickly to a surface compared to PA14 lineages, with early c-di-GMP-mediated exopolysaccharide (EPS) production that can facilitate the attachment of neighbors. PA14 lineages modulate their motility via cyclic AMP (cAMP) and retain memory of the surface so that their progeny are primed for improved subsequent surface attachment. Based on the findings of previous studies, we propose that the differences between PAO1 and PA14 are potentially rooted in downstream differences between Wsp-based and Pil-Chp-based surface-sensing systems, respectively. IMPORTANCE The initial pivotal phase of bacterial biofilm formation known as reversible attachment, where cells undergo a period of transient surface attachment, is at once universal and poorly understood. What is more, although we know that reversible attachment culminates ultimately in irreversible attachment, it is not clear how reversible attachment progresses phenotypically, as bacterial surface-sensing circuits fundamentally alter cellular behavior. We analyze diverse observed bacterial behavior one family at a time (defined as a full lineage of cells related to one another by division) using a unifying stochastic model and show that our findings lead to insights on the time evolution of reversible attachment and the social cooperative dimension of surface attachment in PAO1 and PA14 strains., (Copyright © 2020 Lee et al.)

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration., Competing Interests: CA, CL, JP, Jd, AX, KZ, BT, LH, FJ, CH, GW, MP No competing interests declared, (© 2019, Armbruster et al.)

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections.

Competing Interests: The authors declare no conflict of interest.

Unlabelled: While considerable research has focused on the properties of individual bacteria, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. Staphylococcus aureus frequently coinfects with other pathogens in a range of different infectious diseases. For example, coinfection by S. aureus with Pseudomonas aeruginosa occurs commonly in people with cystic fibrosis and is associated with higher lung disease morbidity and mortality. S. aureus secretes numerous exoproducts that are known to interact with host tissues, influencing inflammatory responses. The abundantly secreted S. aureus staphylococcal protein A (SpA) binds a range of human glycoproteins, immunoglobulins, and other molecules, with diverse effects on the host, including inhibition of phagocytosis of S. aureus cells. However, the potential effects of SpA and other S. aureus exoproducts on coinfecting bacteria have not been explored. Here, we show that S. aureus-secreted products, including SpA, significantly alter two behaviors associated with persistent infection. We found that SpA inhibited biofilm formation by specific P. aeruginosa clinical isolates, and it also inhibited phagocytosis by neutrophils of all isolates tested. Our results indicate that these effects were mediated by binding to at least two P. aeruginosa cell surface structures-type IV pili and the exopolysaccharide Psl-that confer attachment to surfaces and to other bacterial cells. Thus, we found that the role of a well-studied S. aureus exoproduct, SpA, extends well beyond interactions with the host immune system. Secreted SpA alters multiple persistence-associated behaviors of another common microbial community member, likely influencing cocolonization and coinfection with other microbes., Importance: Bacteria rarely exist in isolation, whether on human tissues or in the environment, and they frequently coinfect with other microbes. However, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. We identified a novel interaction between two bacterial species that frequently infect together-Staphylococcus aureus and Pseudomonas aeruginosa We show that the S. aureus-secreted protein staphylococcal protein A (SpA), which is well-known for interacting with host targets, also binds to specific P. aeruginosa cell surface molecules and alters two persistence-associated P. aeruginosa behaviors: biofilm formation and uptake by host immune cells. Because S. aureus frequently precedes P. aeruginosa in chronic infections, these findings reveal how microbial community interactions can impact persistence and host interactions during coinfections., (Copyright © 2016 Armbruster et al.)

Background: Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized., Methods: We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation., Results: Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03-3.83] and 2.14 [1.32-3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial., Conclusions: Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Several bacterial species that are natural inhabitants of potable water distribution system biofilms are opportunistic pathogens important to sensitive patients in healthcare facilities. Waterborne healthcare-associated infections (HAI) may occur during the many uses of potable water in the healthcare environment. Prevention of infection is made more challenging by lack of data on infection rate and gaps in understanding of the ecology, virulence, and infectious dose of these opportunistic pathogens. Some healthcare facilities have been successful in reducing infections by following current water safety guidelines. This review describes several infections, and remediation steps that have been implemented to reduce waterborne HAIs.

Water in healthcare environments can be a source for healthcare-associated infections (HAI). However, information on the exposure risk to opportunistic pathogens in potable water distribution systems (PWDS) is lacking. Laboratory studies characterizing the interaction of opportunistic pathogens with biofilms are needed to understand their role in water systems within healthcare facilities. A stable, repeatable, PWDS multi-species biofilm model comprising Sphingomonas paucimobilis, Methylobacterium sp., Delftia acidovorans, and Mycobacterium mucogenicum was developed in the CDC Biofilm Reactor (CBR), reaching 6 log(10) CFU cm(-2) within 6 days. The model was used to investigate the interaction of the opportunistic pathogen M. mucogenicum with the other species, and to determine the efficacy of monochloramine (NH(2)Cl) as a disinfectant against 2-week-old biofilms. Addition of 1 or 2 mg l(-1) NH(2)Cl resulted in the same or an increased log density of viable M. mucogenicum in the biofilm while inactivating some of the Proteobacteria. Although M. mucogenicum preferentially resided in the biofilm, NH(2)Cl exposure caused release of viable M. mucogenicum from the biofilm into the water. Additional studies with this model should determine if sodium hypochlorite has a comparative effect and if other nontuberculous mycobacteria (NTM) respond to NH(2)Cl similarly.

Background: Healthcare-associated outbreaks and pseudo-outbreaks of rapidly growing mycobacteria (RGM) are frequently associated with contaminated tap water. A pseudo-outbreak of Mycobacterium chelonae-M. abscessus in patients undergoing bronchoscopy was identified by 2 acute care hospitals. RGM was identified in bronchoscopy specimens of 28 patients, 25 of whom resided in the same skilled nursing facility (SNF). An investigation ruled out bronchoscopy procedures, specimen collection, and scope reprocessing at the hospitals as sources of transmission., Objective: To identify the reservoir for RGM within the SNF and evaluate 2 water system treatments, hyperchlorination and point-of-use (POU) membrane filters, to reduce RGM., Design: A comparative in situ study of 2 water system treatments to prevent RGM transmission., Setting: An SNF specializing in care of patients requiring ventilator support., Methods: RGM and heterotrophic plate count (HPC) bacteria were examined in facility water before and after hyperchlorination and in a subsequent 24-week assessment of filtered water by colony enumeration on Middlebrook and R2A media., Results: Mycobacterium chelonae was consistently isolated from the SNF water supply. Hyperchlorination reduced RGM by 1.5 log(10) initially, but the population returned to original levels within 90 days. Concentration of HPC bacteria also decreased temporarily. RGM were reduced below detection level in filtered water, a 3-log(10) reduction. HPC bacteria were not recovered from newly installed filters, although low quantities were found in water from 2-week-old filters., Conclusion: POU membrane filters may be a feasible prevention measure for healthcare facilities to limit exposure of sensitive individuals to RGM in potable water systems.