Cystic Fibrosis Isolates Research Articles

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections.

In this study, we analyzed 15 multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa from chronic lung infections for expression of 4 different multidrug efflux systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY), using quantitative reverse transcriptase PCR. Overexpression of MexXY pump was observed in all of the isolates tested. Analysis of regulatory genes that control the expression of these 4 efflux pumps revealed a number of previously uncharacterized mutations. Our work shows that MexXY pump overexpression is common in cystic fibrosis isolates and could be contributing to their reduced aminoglycoside susceptibility. Further, we also identified novel mutations in the regulatory genes of the 4 abovementioned Resistance-Nodulation-Division superfamily pumps that may be involved in the overexpression of these pumps.

Rifampicin potentiation of aminoglycoside activity against cystic fibrosis isolates of Pseudomonas aeruginosa.

Rifampicin potentiates the activity of aminoglycosides (AGs) versus Pseudomonas aeruginosa by targeting the AmgRS two-component system. In this study we examine the impact of rifampicin on the AG susceptibility of cystic fibrosis (CF) lung isolates of P. aeruginosa and the contribution of AmgRS to AG resistance in these isolates. amgR deletion derivatives of clinical isolates were constructed using standard gene replacement technology. Susceptibility to AGs ± rifampicin (at ½ MIC) was assessed using a serial 2-fold dilution assay. Rifampicin showed a variable ability to potentiate AG activity versus the CF isolates, enhancing AG susceptibility between 2- and 128-fold. Most strains showed potentiation for at least two AGs, with only a few strains showing no AG potentiation by rifampicin. Notably, loss of amgR increased AG susceptibility although rifampicin potentiation of AG activity was still observed in the ΔamgR derivatives. AmgRS contributes to AG resistance in CF isolates of P. aeruginosa and rifampicin shows a variable ability to potentiate AG activity against these, highlighting the complexity of AG resistance in such isolates.

Pseudomonas aeruginosa Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung.

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2 < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

Uncovering Differences in Virulence Markers Associated with Achromobacter Species of CF and Non-CF Origin.

Achromobacter spp. are recognized as emerging pathogens in hospitalized as well as in cystic fibrosis (CF) patients. From 2012 to 2015, we collected 69 clinical isolates (41 patient) of Achromobacter spp. from 13 patients with CF (CF isolates, n = 32) and 28 patients receiving care for other health conditions (non-CF isolates, n = 37). Molecular epidemiology and virulence potential of isolates were examined. Antimicrobial susceptibility, motility, ability to form biofilms and binding affinity to mucin, collagen, and fibronectin were tested to assess their virulence traits. The nrdA gene sequencing showed that A. xylosoxidans was the most prevalent species in both CF and non-CF patients. CF patients were also colonized with A. dolens/A. ruhlandii, A. insuavis, and A. spiritinus strains while non-CF group was somewhat less heterogenous, although A. insuavis, A. insolitus, and A. piechaudii strains were detected beside A. xylosoxidans. Three strains displayed clonal distribution, one among patients from the CF group and two among non-CF patients. No significant differences in susceptibility to antimicrobials were observed between CF and non-CF patients. About one third of the isolates were classified as strong biofilm producers, and the proportion of CF and non-CF isolates with the ability to form biofilm was almost identical. CF isolates were less motile compared to the non-CF group and no correlation was found between swimming phenotype and biofilm formation. On the other hand, CF isolates exhibited higher affinity to bind mucin, collagen, and fibronectin. In generall, CF isolates from our study exhibited in vitro properties that could be of importance for the colonization of CF patients.

Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients.

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth.We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP).Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain.Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients.

We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non-CF patients over a 2 year period. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species-specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non-CF patients, 25 from the hospital environment and three from a commercial company. Potential cross-infection was assessed by pulsed-field gel electrophoresis (PFGE) and multi- locus-sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. Burkholderia multivorans was the most prevalent species among CF patients affecting 56 % (192) patients, followed by Burkholderia cenocepacia IIIA (15 %; 52 patients). Five novel recA clusters were found. Among non-CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34 %), with 18 of 40 isolates part of a UK-wide B. cepacia 'cluster'. This and three other clusters were investigated by PFGE and MLST. Cable-pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non- CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non-CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

In vitro activity of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria.

Diazabicyclooctanes (DBOs) inhibit class A, class C and some class D β-lactamases. A few also bind PBP2, conferring direct antibacterial activity and a β-lactamase-independent 'enhancer' effect, potentiating β-lactams targeting PBP3. We tested a novel DBO, zidebactam, combined with cefepime. CLSI agar dilution MICs were determined with cefepime/zidebactam in a chequerboard format. Bactericidal activity was also measured. Zidebactam MICs were ≤2 mg/L (mostly 0.12-0.5 mg/L) for most Escherichia coli , Klebsiella , Citrobacter and Enterobacter spp., but were >32 mg/L for Proteeae, most Serratia and a few E. coli , Klebsiella and Enterobacter/Citrobacter . The antibacterial activity of zidebactam dominated chequerboard studies for Enterobacteriaceae, but potentiation of cefepime was apparent for zidebactam-resistant isolates with class A and C enzymes, illustrating β-lactamase inhibition. Overall, cefepime/zidebactam inhibited almost all Enterobacteriaceae with AmpC, ESBL, K1, KPC and OXA-48-like β-lactamases at 1 + 1 mg/L and also 29 of 35 isolates with metallo-carbapenemases, including several resistant to zidebactam alone. Zidebactam MICs for 36 of 50 Pseudomonas aeruginosa were 4-16 mg/L, and the majority of AmpC, metallo-β-lactamase-producing and cystic fibrosis isolates were susceptible to cefepime/zidebactam at 8 + 8 mg/L. Zidebactam MICs for Acinetobacter baumannii and Stenotrophomonas maltophilia were >32 mg/L; potentiation of cefepime was frequent for S. maltophilia , but minimal for A. baumannii . Kill curve results largely supported MICs. Zidebactam represents a second triple-action DBO following RG6080, with lower MICs for Enterobacteriaceae and P. aeruginosa . Clinical evaluation of cefepime/zidebactam must critically evaluate the reliance that can be placed on this direct antibacterial activity and on the enhancer effect as well as β-lactamase inhibition.

Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic

The β-lactam antibiotic temocillin (6-α-methoxy-ticarcillin) shows stability to most extended spectrum β-lactamases, but is considered inactive against Pseudomonas aeruginosa. Mutations in the MexAB-OprM efflux system, naturally occurring in cystic fibrosis (CF) isolates, have been previously shown to reverse this intrinsic resistance. In the present study, we measured temocillin activity in a large collection (n = 333) of P. aeruginosa CF isolates. 29% of the isolates had MICs ≤ 16 mg/L (proposed clinical breakpoint for temocillin). Mutations were observed in mexA or mexB in isolates for which temocillin MIC was ≤512 mg/L (nucleotide insertions or deletions, premature termination, tandem repeat, nonstop, and missense mutations). A correlation was observed between temocillin MICs and efflux rate of N-phenyl-1-naphthylamine (MexAB-OprM fluorescent substrate) and extracellular exopolysaccharide abundance (contributing to a mucoid phenotype). OpdK or OpdF anion-specific porins expression decreased temocillin MIC by ~1 two-fold dilution only. Contrarily to the common assumption that temocillin is inactive on P. aeruginosa, we show here clinically-exploitable MICs on a non-negligible proportion of CF isolates, explained by a wide diversity of mutations in mexA and/or mexB. In a broader context, this work contributes to increase our understanding of MexAB-OprM functionality and help delineating how antibiotics interact with MexA and MexB.

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates.

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 μg/ml reduced completely 24 h old biofilms while concentration of 25 μg/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated.

Outbreak of Hospital Infection from Biofilm-embedded Pan Drug-resistant Pseudomonas aeroginosa, Due to a Contaminated Bronchoscope.

Colistin-resistant Pseudomonas aeruginosa (P. aeruginosa) has been defined as pandrug-resistant (PDR) strain. Outbreaks of PDR P. aeruginosa especially in pulmonary tract infections due to contaminated bronchoscopes have rarely been reported. The emergence of pandrug-resistant strains in both CF (Cystic Fibrosis) and non-CF clinical isolates over recent years remains of a great concern. Hospital wards contaminated with PDR P. aeruginosa infection, must be shot down until their eradication. Health Authorities must be informed immediately and infection control strategies must be implemented. To report such an outbreak and modify the infection control strategy in an academic hospital in Ankara Turkey. From October to December 2013, PDR-Pseudomonas aerogionsa were identified from bronchial cultures of 15 patients who had undergone bronchoscopy prior to the infection. Three batches of surveillance cultures were obtained from the environmental objects and healthcare workers related to the procedures. Pulsed-field gel electrophoresis (PFGE) was used for bacterial typing. Antimicrobial susceptibility was assessed by disc diffusion and E-test methods. A total of 70 specimens were obtained during the first surveillance operation. One Colistin-resistant P. aeroginosa was isolated from a bronchoscope. Although the disinfection protocols for bronchoscope were revised and implemented, seven additional bronchial cases were identified thereafter. The pathogen was identified from two subsequent surveillance cultures and was not eliminated until Ethylene oxide sterilization was added to the disinfection protocol. PFGE revealed that all 15 isolates from the patients and the three isolates from the bronchoscope shared a common pattern with minor variance. XbaI restriction enzyme turned out better than SpeI in interpreting bacterial pulse types with BioNumerics 6.0. The most suitable cut off value for SpeI was above 80% Dice similarity while for XbaI above 95%Dice similarity with BioNumerics 6.0. The outbreak of "Colistin" pan drug-resistant Pseudomonas aeroginosa was caused by a contaminated bronchoscope and was terminated by the implementation of a revised disinfection protocol for bronchoscope.

Virulence adaptations of Pseudomonas aeruginosa isolated from patients with non-cystic fibrosis bronchiectasis.

Pseudomonas aeruginosa is a major pathogen in chronic lung diseases such as cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis (nCFB). Much of our understanding regarding infections in nCFB patients is extrapolated from findings in CF with little direct investigation on the adaptation of P. aeruginosa in nCFB patients. As such, we investigated whether the adaptation of P. aeruginosa was indeed similar between nCFB and CF. From our prospectively collected biobank, we identified 40 nCFB patients who had repeated P. aeruginosa isolates separated by ≥6 months and compared these to a control population of 28 CF patients. A total of 84 nCFB isolates [40 early (defined as the earliest isolate in the biobank) and 41 late (defined as the last available isolate in the biobank)] were compared to 83 CF isolates (39 early and 44 late). We assessed the isolates for protease, lipase and elastase production; mucoid phenotype; swarm and swim motility; biofilm production; and the presence of the lasR mutant phenotype. Overall, we observed phenotypic heterogeneity in both nCFB and CF isolates and found that P. aeruginosa adapted to the nCFB lung environment similarly to the way observed in CF isolates in terms of protease and elastase expression, motility and biofilm formation. However, significant differences between nCFB and CF isolates were observed in lipase expression, which may allude to distinct characteristics found in the lung environment of nCFB patients. We also sought to determine virulence potential over time in nCFB P. aeruginosa isolates and found that virulence decreased over time, similar to CF.

Genotypic and Phenotypic Characterization of Stenotrophomonas maltophilia Strains from a Pediatric Tertiary Care Hospital in Serbia.

BackgroundStenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients.MethodsWe characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results.ResultsMost of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed.ConclusionsHigh genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.

High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations

High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine–glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg β-naphthylamide (PAβN). β-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64–128/16–32 mg/L and 128/128–256 mg/L in CF and HAP isolates, respectively; PAβN reduced meropenem MICs to 4–16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted.

LasR Variant Cystic Fibrosis Isolates Reveal an Adaptable Quorum-Sensing Hierarchy in Pseudomonas aeruginosa.

ABSTRACTChronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR.

An ex vivo lung model to study bronchioles infected with Pseudomonas aeruginosa biofilms.

A key aim in microbiology is to determine the genetic and phenotypic bases of bacterial virulence, persistence and antimicrobial resistance in chronic biofilm infections. This requires tractable, high-throughput models that reflect the physical and chemical environment encountered in specific infection contexts. Such models will increase the predictive power of microbiological experiments and provide platforms for enhanced testing of novel antibacterial or antivirulence therapies. We present an optimized ex vivo model of cystic fibrosis lung infection: ex vivo culture of pig bronchiolar tissue in artificial cystic fibrosis mucus. We focus on the formation of biofilms by Pseudomonas aeruginosa. We show highly repeatable and specific formation of biofilms that resemble clinical biofilms by a commonly studied laboratory strain and ten cystic fibrosis isolates of this key opportunistic pathogen.

RRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus.

The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community.

Surfactant Protein A Recognizes Outer Membrane Protein OprH on Pseudomonas aeruginosa Isolates From Individuals With Chronic Infection.

Surfactant protein A (SP-A) plays a critical role in the clearance of Pseudomonas aeruginosa from the lung. However, there is limited information about the interaction of this protein with P. aeruginosa isolates from individuals with cystic fibrosis (CF). We characterized the interplay between SP-A and a collection of isogenic sequential isolates from 7 patients with CF. We identified outer membrane protein OprH as a novel ligand for SP-A on P. aeruginosa The last-available (late) isolates from patients with CF bound significantly less SP-A than their respective first-available (early) isolates. This difference could be associated with a reduction in the expression of OprH. Binding of SP-A to OprH promoted phagocytic killing; thus, late CF isolates were at least 2-fold more resistant to SP-A-mediated killing by human macrophages than their respective early isolates. We postulate that the reduction of OprH expression is a previously unrecognized adaptation of P. aeruginosa to the lung of individuals with CF that facilitates the escape of the microorganism from SP-A-mediated phagocytic killing.

Activity of Cysteamine against the Cystic Fibrosis Pathogen Burkholderia cepacia Complex

There are no wholly successful chemotherapeutic strategies against Burkholderia cepacia complex (BCC) colonization in cystic fibrosis (CF). We assessed the impact of cysteamine (Lynovex) in combination with standard-of-care CF antibiotics in vitro against BCC CF isolates by the concentration at which 100% of bacteria were killed (MIC100) and checkerboard assays under CLSI standard conditions. Cysteamine facilitated the aminoglycoside-, fluoroquinolone- and folate pathway inhibitor-mediated killing of BCC organisms that were otherwise resistant or intermediately sensitive to these antibiotic classes. Slow-growing BCC strains are often recalcitrant to treatment and form biofilms. In assessing the impact of cysteamine on biofilms, we demonstrated inhibition of BCC biofilm formation at sub-MIC100s of cysteamine.