Pathogen In Cystic Fibrosis Patients Research Articles

Mycobacterium abscessus Opsonization Allows an Escape from the Defensin Bactericidal Action in Drosophila.

Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in Drosophila that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by Drosophila macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both in vitro and in vivo and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in Drosophila. IMPORTANCE Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using Drosophila, we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by Drosophila phagocytes. Indeed, by combining in vivo and in vitro approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.

Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Antitoxin CrlA of CrlTA Toxin-Antitoxin System in a Clinical Isolate Pseudomonas aeruginosa Inhibits Lytic Phage Infection.

Pseudomonas aeruginosa is an important opportunistic pathogen in cystic fibrosis patients and immunocompromised individuals, and the toxin–antitoxin (TA) system is involved in bacterial virulence and phage resistance. However, the roles of TA systems in P. aeruginosa are relatively less studied and no phage Cro-like regulators were identified as TA components. Here, we identified and characterized a chromosome-encoded prophage Cro-like antitoxin (CrlA) in the clinical isolate P. aeruginosa WK172. CrlA neutralized the toxicity of the toxin CrlA (CrlT) which cleaves mRNA, and they formed a type II TA system. Specifically, crlA and crlT are co-transcribed and their protein products interact with each other directly. The autorepression of CrlA is abolished by CrlT through the formation of the CrlTA complex. Furthermore, crlTA is induced in the stationary phase, and crlA is expressed at higher levels than crlT. The excess CrlA inhibits the infection of lytic Pseudomonas phages. CrlA is widely distributed among Pseudomonas and in other bacterial strains and may provide antiphage activities.

Genomic Analysis of Endophytic Bacillus-Related Strains Isolated from the Medicinal Plant Origanum vulgare L. Revealed the Presence of Metabolic Pathways Involved in the Biosynthesis of Bioactive Compounds.

Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant Origanum vulgare L. and able to antagonize the growth of opportunistic pathogens of cystic fibrosis patients. The in silico analysis revealed the presence of gene clusters involved in the production of antimicrobial compounds, such as paeninodin, paenilarvins, polymyxin, and paenicidin A. Endophytes’ adaptation to the plant microenvironment was evaluated through the analysis of the presence of antibiotic resistance genes in the four genomes. The diesel fuel degrading potential was also tested. Strains grew in minimum media supplemented with diesel fuel, but no n-alkanes degradation genes were found in their genomes, suggesting that diesel fuel degradation might occur through other steps involving enzymes catalyzing the oxidation of aromatic compounds.

Viable but non-cultivable state: a strategy for Staphylococcus aureus survivable in dual-species biofilms with Pseudomonas aeruginosa?

Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in cystic fibrosis patients. Both organisms often cause chronic and recalcitrant infections, in large part due to their ability to form biofilms, being these mixed-species infections correlated with poor clinical outcomes. In this study, the hypothesis that S. aureus adopts phenotypes allowing its coexistence with P. aeruginosa during biofilm growth was put forward. We noticed that S. aureus undergoes a viable but non-cultivable (VBNC) state in the dominated P. aeruginosa dual-species consortia, whatsoever the strains used to form the biofilms. Moreover, an increased expression of genes associated with S. aureus virulence was detected suggesting that the phenotypic switching to VBNC state might account for S. aureus pathogenicity and, in turn, influence the clinical outcome of the mixed-species infection. Thus, P. aeruginosa seems to induce both phenotypic and transcriptomic changes in S. aureus, helping its survival and coexistence in the dual-species biofilms. Overall, our findings illustrate how interspecies interactions can modulate bacterial virulence in vitro, contributing to a better understanding of the behaviour of P. aeruginosa-S. aureus dual-species biofilms.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

Fluoroquinolone resistance in Achromobacter spp.: substitutions in QRDRs of GyrA, GyrB, ParC and ParE and implication of the RND efflux system AxyEF-OprN.

Achromobacter are emerging pathogens in cystic fibrosis patients. Mechanisms of resistance to fluoroquinolones are unknown in clinical isolates. Among non-fermenting Gram-negative bacilli, fluoroquinolone resistance is mostly due to amino acid substitutions in localized regions of the targets (GyrA, GyrB, ParC and ParE) named QRDRs, but also to efflux. To explore quinolone resistance mechanisms in Achromobacter. The putative QRDRs of GyrA, GyrB, ParC and ParE were sequenced in 62 clinical isolates, and in vitro one-step mutants obtained after exposure to fluoroquinolones. An in vitro mutant and its parental isolate were investigated by RNASeq and WGS. RT-qPCR and gene inactivation were used to explore the role of efflux systems overexpression. We detected seven substitutions in QRDRs (Q83L/S84P/D87N/D87G for GyrA, Q480P for GyrB, T395A/K525Q for ParE), all in nine of the 27 clinical isolates with ciprofloxacin MIC ≥16 mg/L, whereas none among the in vitro mutants. The RND efflux system AxyEF-OprN was overproduced (about 150-fold) in the in vitro mutant NCF-39-Bl6 versus its parental strain NCF-39 (ciprofloxacin MICs 64 and 1.5 mg/L, respectively). A substitution in AxyT (putative regulator of AxyEF-OprN) was detected in NCF-39-Bl6. Ciprofloxacin MIC in NCF-39-Bl6 dropped from 64 to 1.5 mg/L following gene inactivation of either axyT or axyF. Substitutions in AxyT associated with overexpression of AxyEF-OprN were also detected in seven clinical strains with ciprofloxacin MIC ≥16 mg/L. Target alteration is not the primary mechanism involved in fluoroquinolone resistance in Achromobacter. The role of AxyEF-OprN overproduction was demonstrated in one in vitro mutant.

P114 The analysis of bacterial pathogens in cystic fibrosis patients of the Russian Federation in 2018–2019 with evaluation of antibiotic resistance

P114 The analysis of bacterial pathogens in cystic fibrosis patients of the Russian Federation in 2018–2019 with evaluation of antibiotic resistance

P049 The epidemiology of bacterial pathogens in cystic fibrosis patients from the Russian Federation

P049 The epidemiology of bacterial pathogens in cystic fibrosis patients from the Russian Federation

Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh.

Burkholderia cepacia complex (Bcc) is a group of serious pathogens in cystic fibrosis patients and causes life threatening infections in immunocompromised patients. Species within the Bcc are widely distributed within the environment, can survive in the presence of disinfectants and antiseptics, and are inherently multidrug resistant (MDR). Dhaka Medical College Hospital (DMCH) patients with a B. cepacia positive blood culture between 20 October 2016 to 23rd September 2017 were considered as outbreak cases. Blood stream infections (BSIs) were detected using BacT/ALERT 3D at DMCH. B. cepacia was isolated on chromogenic UTI media followed by MALDI-TOF. Minimum inhibitory concentration (MIC) of clinically relevant antibiotics was determined by agar dilution. Whole genome sequencing was performed on an Illumina MiSeq platform. Patients' demographic and clinical data were collected. Patients' clinical history and genomic data of the outbreak strains were merged to investigate possible outbreaks. Ninety-one B. cepacia genomes were downloaded from 'Burkholderia Genome Database' and the genomic background of the global strains were compared with our outbreak strains. Among 236 BSIs, 6.35% (15/236) were B. cepacia. Outbreak cases were confined to the burn critical care unit and, to a lesser extent, the paediatrics department. There was a continuum of overlapping cases at DMCH between 23 October 2016 to 30 August 2017. Core genome SNPs showed that the outbreak strains were confined to a single clade, corresponded to a common clone (ST1578). The strains were shown to be MDR and associated with a mortality of 31% excluding discharge against medical advice. MIC profiles of the strains suggested that antibiotics deployed as empirical therapy were invariably inappropriate. The genetic background of the outbreak strains was very similar; however, a few variations were found regarding the presence of virulence genes. Compared to global strains from the Burkholderia Genome Database, the Bangladeshi strains were genetically distinct. Environmental surveillance is required to investigate the aetiology and mode of transmission of the B. cepacia outbreak. Systematic management of nosocomial outbreaks, particularly in resource limited regions, will mitigate transmission and will improve patients' outcomes.

Comparative Genomics and Evolutionary Analysis of RNA-Binding Proteins of Burkholderia cenocepacia J2315 and Other Members of the B. cepacia Complex.

RNA-binding proteins (RBPs) are important regulators of cellular functions, playing critical roles on the survival of bacteria and in the case of pathogens, on their interaction with the host. RBPs are involved in transcriptional, post-transcriptional, and translational processes. However, except for model organisms like Escherichia coli, there is little information about the identification or characterization of RBPs in other bacteria, namely in members of the Burkholderia cepacia complex (Bcc). Bcc is a group of bacterial species associated with a poor clinical prognosis in cystic fibrosis patients. These species have some of the largest bacterial genomes, and except for the presence of two-distinct Hfq-like proteins, their RBP repertoire has not been analyzed so far. Using in silico approaches, we identified 186 conventional putative RBPs in Burkholderia cenocepacia J2315, an epidemic and multidrug resistant pathogen of cystic fibrosis patients. Here we describe the comparative genomics and phylogenetic analysis of RBPs present in multiple copies and predicted to play a role in transcription, protein synthesis, and RNA decay in Bcc bacteria. In addition to the two different Hfq chaperones, five cold shock proteins phylogenetically close to E. coli CspD protein and three distinct RhlE-like helicases could be found in the B. cenocepacia J2315 genome. No RhlB, SrmB, or DeaD helicases could be found in the genomes of these bacteria. These results, together with the multiple copies of other proteins generally involved in RNA degradation, suggest the existence, in B. cenocepacia and in other Bcc bacteria, of some extra and unexplored functions for the mentioned RBPs, as well as of alternative mechanisms involved in RNA regulation and metabolism in these bacteria.

Clinical relevance of Scedosporium spp. and Exophiala dermatitidis in patients with cystic fibrosis: A nationwide study.

An increased prevalence of various filamentous fungi in sputum samples of patients with cystic fibrosis (CF) has been reported. The clinical significance, however, is mostly unclear. The aim of this study was to investigate the clinical relevance of Scedosporium spp. and Exophiala dermatitidis from sputum samples of patients with CF in the Netherlands. In this cross-sectional study, all CF patients of the Dutch national CF registry who were treated at five of the seven recognized CF centers during a 3-year period were included. We linked clinical data of the national CF registry with the national Dutch filamentous fungal database. We investigated the association between clinical characteristics and a positive sputum sample for Scedosporium spp. and E. dermatitidis, using logistic regression. Positive cultures for fungi were obtained from 3787 sputum samples from 699 of the 1312 patients with CF. Scedosporium spp. was associated with severe genotype, CF-related diabetes, several microorganisms, and inhaled antibiotics. E. dermatitidis was associated with older age, female sex, and Aspergillus spp. CF patients with and without Scedosporium spp. or E. dermatitidis seemed comparable in body mass index and lung function. This study suggests that Scedosporium spp. and E. dermatitidis are probably no major pathogens in CF patients in the Netherlands. Greater understanding of epidemiologic trends, risk factors, and pathogenicity of filamentous fungi in the respiratory tracts of patients with CF is needed.

Nanomedicine Approaches for the Pulmonary Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disease affecting today nearly 70,000 patients worldwide and characterized by a hypersecretion of thick mucus difficult to clear arising from the defective CFTR protein. The over-production of the mucus secreted in the lungs, along with its altered composition and consistency, results in airway obstruction that makes the lungs susceptible to recurrent and persistent bacterial infections and endobronchial chronic inflammation, which are considered the primary cause of bronchiectasis, respiratory failure, and consequent death of patients. Despite the difficulty of treating the continuous infections caused by pathogens in CF patients, various strategies focused on the symptomatic therapy have been developed during the last few decades, showing significant positive impact on prognosis. Moreover, nowadays, the discovery of CFTR modulators as well as the development of gene therapy have provided new opportunity to treat CF. However, the lack of effective methods for delivery and especially targeted delivery of therapeutics specifically to lung tissues and cells limits the efficiency of the treatments. Nanomedicine represents an extraordinary opportunity for the improvement of current therapies and for the development of innovative treatment options for CF previously considered hard or impossible to treat. Due to the peculiar environment in which the therapies have to operate characterized by several biological barriers (pulmonary tract, mucus, epithelia, bacterial biofilm) the use of nanotechnologies to improve and enhance drug delivery or gene therapies is an extremely promising way to be pursued. The aim of this review is to revise the currently used treatments and to outline the most recent progresses about the use of nanotechnology for the management of CF.

2663. Impact of Pre-Transplant Microbiology on Acute Outcomes in Cystic Fibrosis Patients Receiving Bilateral Lung Transplants

BackgroundLung transplantation is a life-prolonging intervention for cystic fibrosis (CF) patients; however, their tendency to be colonized with multiple respiratory pathogens poses a unique risk for post-transplant complications. While infections with certain CF-related pathogens have been identified as contraindications for transplant, much remains uncertain about the influence of pre-transplant microbiological factors on post-transplant outcomes.MethodsA retrospective cohort study was performed for all CF patients receiving bilateral lung transplants at a single center during the 2016–2018 period. Patient and microbiological data were collected and analyzed from 1 year pre-transplant to 3 months post-transplant. Patients were categorized according to pre-transplant microbiology, with consideration to multidrug-resistant organisms (MDROs) and chronic organisms (positive culture in ≥ 50% of encounters).ResultsTwenty-seven CF patients received a transplant during this time period. Twenty-five patients (92.6%) had re-isolation with ≥ 1 pre-transplant organism in the 3 month period post-transplant, with 16 (59.3%) developing infectious complications, and 11 (40.7%) developing rejection. Isolates associated with chronic infections were the principal factor in determining re-isolation post-transplant (OR = 4.353, 95% CI = 1.455–13.027, P = 0.009). Multidrug-resistance (P = 0.095) and species (P > 0.3) were not significant predictors of re-isolation. There was no difference in early post-transplant outcomes (infectious complications, rejection, FEV1% predicted, ICU and hospital LOS) for patients chronically infected with MDROs vs. those who were not (P > 0.3). Chronic infections with Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus were not predictors of poor outcomes (P > 0.3). However, chronic fungal infections (n = 7) produced more infectious complications (median 2 vs. 0, P = 0.0453) and longer ICU stays (median 22 days vs. 5 days, P = 0.0191).ConclusionChronic infections are associated with a greater risk of post-transplant re-isolation of pathogens in CF patients, more so than drug resistance or species. Chronic infections with fungi were associated with worse transplant outcomes.Disclosures All authors: No reported disclosures.

Pneumonia due to Pandoraea Apista after evacuation of traumatic intracranial hematomas:a case report and literature review

BackgroundPandoraea species is a newly described genus, which is multidrug resistant and difficult to identify. Clinical isolates are mostly cultured from cystic fibrosis (CF) patients. CF is a rare disease in China, which makes Pandoraea a total stranger to Chinese physicians. Pandoraea genus is reported as an emerging pathogen in CF patients in most cases. However, there are few pieces of evidence that confirm Pandoraea can be more virulent in non-CF patients. The pathogenicity of Pandoraea genus is poorly understood, as well as its treatment. The incidence of Pandoraea induced infection in non-CF patients may be underestimated and it’s important to identify and understand these organisms.Case presentationWe report a 44-years-old man who suffered from pneumonia and died eventually. Before his condition deteriorated, a Gram-negative bacilli was cultured from his sputum and identified as Pandoraea Apista by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS).ConclusionPandoraea spp. is an emerging opportunistic pathogen. The incidences of Pandoraea related infection in non-CF patients may be underestimated due to the difficulty of identification. All strains of Pandoraea show multi-drug resistance and highly variable susceptibility. To better treatment, species-level identification and antibiotic susceptibility test are necessary.

Chronic Airway Colonization by Achromobacter xylosoxidans in Cystic Fibrosis Patients Is Not Sustained by Their Domestic Environment.

Achromobacter spp. are nonfermentative Gram-negative bacilli considered emergent pathogens in cystic fibrosis (CF). Although some cross-transmission events between CF patients have been described, Achromobacter strains were mostly patient specific, suggesting sporadic acquisitions from nonhuman reservoirs. However, sources of these emergent CF pathogens remain unknown. A large collection of specimens (n = 273) was sampled in the homes of 3 CF patients chronically colonized by Achromobacter xylosoxidans with the aim of evaluating the potential role of domestic reservoirs in sustaining airway colonization of the patients. Samples were screened for the presence of Achromobacter by using genus-specific molecular detection. Species identification, multilocus genotypes, and antimicrobial susceptibility patterns observed for environmental isolates were compared with those of clinical strains. Patient homes hosted a high diversity of Achromobacter species (n = 7), including Achromobacter mucicolens and A. animicus, two species previously isolated from human samples only, and genotypes (n = 15), all showing an overall susceptibility to antimicrobial agents. Achromobacter strains were mostly isolated from indoor moist environments and siphons, which are potential reservoirs for several CF emerging pathogens. A. xylosoxidans, the worldwide prevalent species colonizing CF patients, was not the major Achromobacter species inhabiting domestic environments. A. xylosoxidans genotypes chronically colonizing the patients were not detected in their household environments. These results support the notions that the domestic environment could not be incriminated in sustained patient colonization and that after initial colonization, the environmental survival of A. xylosoxidans clones adapted to the CF airways is probably impaired.IMPORTANCEAchromobacter spp. are worldwide emerging opportunistic pathogens in CF patients, able to chronically colonize the respiratory tract. Apart from regular consultations at the hospital CF center, patients spend most of their time at home. Colonization from nonhuman sources has been suggested, but the presence of Achromobacter spp. in CF patients' homes has not been explored. The domestic environments of CF patients chronically colonized by Achromobacter, especially wet environments, host several opportunistic pathogens, including a large diversity of Achromobacter species and genotypes. However, Achromobacter genotypes colonizing the patients were not detected in their domestic environments, making it unlikely that a shuttle between environment and CF airways is involved in persisting colonization. This also suggests that once the bacteria have adapted to the respiratory tract, their survival in the domestic environment is presumably impaired. Nevertheless, measures for reducing domestic patient exposure should be targeted on evacuation drains, which are frequently contaminated by CF opportunistic pathogens.

The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315.

Burkholderia cenocepacia J2315 is a member of the B.cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B.cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B.cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B.cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B.cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to "cepacia syndrome," a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B.cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B.cenocepacia that are relevant for infection.

Design and Synthesis of 4-Alkylidene-β-lactams: Benzyl- and Phenethyl-carbamates as Key Fragments to Switch on Antibacterial Activity.

The emergence of multidrug-resistant bacterial strains is particularly important in chronic pathologies such as cystic fibrosis (CF), in which persistent colonization and selection of resistant strains is favored by the frequent and repeated use of antibacterial agents. Staphylococcus aureus is a common pathogen in CF patients that has an associated increased multidrug resistance. In previous studies we demonstrated that the presence of a 4-alkylidene side chain directly linked to a β-lactam appeared to strengthen the potency against S. aureus, especially against methicillin-resistant S. aureus (MRSA) strains. In the present study, 21 new 4-alkylidene-β-lactams were synthesized and evaluated for antibacterial activity. We designed the new compounds to have aryl, benzyl, or phenethyl-carbamate groups on the C3 hydroxyethyl side chain. We found a correlation between biological activity and the nitrogen substituent of the carbamate group, and two phenethyl-carbamate β-lactams were shown to be valuable antibacterial agents against selected linezolid-resistant strains, with a minimum inhibitory concentrations of 2-4 mg L-1 .

Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection.

ABSTRACTThe airways of cystic fibrosis (CF) patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a component of the P. aeruginosa Pseudomonas quinolone signal (PQS) system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.

Microbiology of the Upper and Lower Airways in Pediatric Cystic Fibrosis Patients.

Objective To evaluate the microbiology of the upper and lower airways in pediatric cystic fibrosis (CF) patients who underwent sinus surgery. Study Design Retrospective case series with chart review. Setting Tertiary care children's hospital. Subjects and Methods A total of 201 paired sinus and pulmonary cultures from 105 CF patients were identified between 1996 and 2014. Demographics and culture results were analyzed. Results The mean age of patients was 11.2 ± 5.4 years (range, 1-27 years), and approximately one-half were female. Methicillin-sensitive Staphylococcus aureus was the most common pathogen overall. A significantly higher prevalence of Pseudomonas aeruginosa (32% for pulmonary and 37% for sinus cultures) was observed in older patients versus younger patients ( P < .001). There was low to moderate agreement between sinus and pulmonary cultures (Kappa statistic range, 0.03-0.56). The prevalence of methicillin-resistant S aureus (MRSA) increased significantly for lower respiratory tract culture (from 5% to 16%) and sinus culture (from 5% to 27%) between 1996-2004 and 2010-2014 ( P = .016 and P < .001, respectively). The prevalence of positive sinus cultures increased from 40% to 85% between 1996-2004 and 2010-2014 ( P = .018). Patients with pulmonary MRSA were more likely to be coinfected with pulmonary P aeruginosa (risk ratio, 2.4; 95% CI, 1.2-4.8; P = .015) or Aspergillus fumigatus (risk ratio, 2.2; 95% CI, 1.2-4.8; P = .035). Conclusions There is low to moderate correlation between pulmonary and sinus pathogens in CF patients. This is important to consider when treating infections. The prevalence of MRSA in sinus cultures has increased over time and warrants further investigation.