Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection in children with cystic fibrosis (SCVSA): a multicentre, observational study.

BACKGROUND: Staphylococcus aureus is the bacterium cultured most often from respiratory secretions of people with cystic fibrosis. Both meticillin-susceptible S aureus and meticillin-resistant S aureus (MRSA) can adapt to form slow-growing, antibiotic-resistant isolates known as small-colony variants that are not routinely identified by clinical laboratories. We aimed to determine the prevalence and clinical significance of S aureus small-colony variants and their subtypes among children with cystic fibrosis. METHODS: The Small Colony Variant Staphylococcus aureus (SCVSA) study was a 2-year longitudinal study of children aged 6-16 years at five US cystic fibrosis centres, using culture methods sensitive for small-colony variants. Children were eligible if they had a documented diagnosis of cystic fibrosis and a minimum of two cystic fibrosis clinic visits and two respiratory cultures in the previous 12 months at enrolment. Participants attended clinic visits quarterly, at which respiratory tract samples were taken and measures of lung function (percentage of predicted forced expiratory volume in 1 s [FEV1] and frequency of respiratory exacerbations) were recorded. We determined the prevalence of small-colony variants and their subtypes, and assessed their independent associations with lung function and respiratory exacerbations using linear mixed-effects and generalised estimating equation logistic regression models. Analyses included both univariate models (unadjusted) and multivariate models that adjusted for potential confounders, including age, sex, race, baseline microbiology, treatment with CFTR modulator, and CTFR genotype. FINDINGS: Between July 1, 2014, and May 26, 2015, we enrolled 230 children. Participants were followed-up for 2 years, with a mean of 6·4 visits (SD 1·14) per participant (range 2-9 visits) and a mean interval between visits of 3·94 months (SD 1·77). Across the 2-year period, S aureus small-colony variants were detected in 64 (28%) participants. Most (103 [56%] of 185) of the small-colony variants detected in these participants were thymidine dependent. Children with small-colony variants had significantly lower mean percentage of predicted FEV1 at baseline than did children without small-colony variants (85·5 [SD 19] vs 92·4 [SD 18·6]; p=0·0145). Small-colony variants were associated with significantly lower percentage of predicted FEV1 throughout the study in regression models, both in univariate analyses (regression coefficient -7·07, 95% CI -12·20 to -1·95; p=0·0068) and in multivariate analyses adjusting for potential confounders (-5·50, -10·51 to -0·48; p=0·0316). Small colony variants of the thymidine-dependent subtype had the strongest association with lung function in multivariate regression models (regression coefficient -10·49, -17·25 to -3·73; p=0·0024). Compared with children without small-colony variants, those with small-colony variants had significantly increased odds of respiratory exacerbations in univariate analyses (odds ratio 1·73, 95% CI 1·19 to 2·52; p=0·0045). Children with thymidine-dependent small-colony variants had significantly increased odds of respiratory exacerbations (2·81, 1·69-4·67; p=0·0001), even after adjusting for age, sex, race, genotype, CFTR modulator, P aeruginosa culture status, and baseline percentage of predicted FEV1 (2·17, 1·33-3·57; p=0·0021), whereas those with non-thymidine-dependent small-colony variants did not. In multivariate models including small-colony variants and MRSA status, P aeruginosa was not independently associated with lung function (regression coefficient -4·77, 95% CI -10·36 to 0·83; p=0·10) and was associated with reduced odds of exacerbations (0·54, 0·36 to 0·81; p=0·0028). Only the small-colony variant form of MRSA was associated with reduced lung function (-8·44, -16·15 to -0·72; p=0·0318) and increased odds of exacerbations (2·15, 1·24 to 3·71; p=0·0061). INTERPRETATION: Infection with small-colony variants, and particularly thymidine-dependent small-colony variants, was common in a multicentre paediatric population with cystic fibrosis and associated with reduced lung function and increased risk of respiratory exacerbations. The adoption of small-colony variant identification and subtyping methods by clinical laboratories, and the inclusion of small-colony variant prevalence data in cystic fibrosis registries, should be considered for ongoing surveillance and study. FUNDING: The Cystic Fibrosis Foundation and the National Institutes of Health.

Optimized In Vitro Antibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus.

Staphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Cystic fibrosis-adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses.

Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease- dependent cytokine degradation. In subacute pulmonary infections, lasR mutant-infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

In Vitro Antibiotic Susceptibility of Initial Pseudomonas aeruginosa Isolates From United States Cystic Fibrosis Patients.

Chronic antibiotic suppression of cystic fibrosis (CF) infections has increased in the United States, as has isolation of Pseudomonas aeruginosa with reduced antibiotic susceptibility. However, analysis of the susceptibilities of 193 initial P aeruginosa clinical isolates suggests that antibiotic susceptibilities are comparable with wild-type strains despite expanded antipseudomonal treatment in the CF community.

Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens.

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings.

Pseudomonas aeruginosa in cystic fibrosis patients with G551D-CFTR treated with ivacaftor.

BACKGROUND: Ivacaftor improves outcomes in cystic fibrosis (CF) patients with the G551D mutation; however, effects on respiratory microbiology are largely unknown. This study examines changes in CF respiratory pathogens with ivacaftor and correlates them with baseline characteristics and clinical response. METHODS: The G551D Observational Study enrolled a longitudinal observational cohort of US patients with CF aged 6 years and older with at least 1 copy of the G551D mutation. Results were linked with retrospective and prospective culture data in the US Cystic Fibrosis Foundation's National Patient Registry. Pseudomonas aeruginosa infection category in the year before and year after ivacaftor was compared and correlated with clinical findings. RESULTS: Among 151 participants prescribed ivacaftor, 29% (26/89) who were culture positive for P. aeruginosa the year prior to ivacaftor use were culture negative the year following treatment; 88% (52/59) of those P. aeruginosa free remained uninfected. The odds of P. aeruginosa positivity in the year after ivacaftor compared with the year prior were reduced by 35% (odds ratio [OR], 0.65; P < .001). Ivacaftor was also associated with reduced odds of mucoid P. aeruginosa (OR, 0.77; P = .013) and Aspergillus (OR, 0.47; P = .039), but not Staphylococcus aureus or other common CF pathogens. Patients with intermittent culture positivity and higher forced expiratory volume in 1 second (FEV1) were most likely to turn culture negative. Reduction in P. aeruginosa was not associated with change in FEV1, body mass index, or hospitalizations. CONCLUSIONS: Pseudomonas aeruginosa culture positivity was significantly reduced following ivacaftor treatment. Efficacious CFTR modulation may contribute to lower frequency of culture positivity for P. aeruginosa and other respiratory pathogens, particularly in patients with less established disease.

Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells.

Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic-resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, whereas the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects under other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain "poisons," can be safely nebulized at high concentration in humans.

Enhanced in vitro formation and antibiotic resistance of nonattached Pseudomonas aeruginosa aggregates through incorporation of neutrophil products.

Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed to P. aeruginosa from the natural environment, and by adulthood, 80% of patients are chronically infected. P. aeruginosa in the CF airway exhibits a unique biofilm-like structure, where it grows in small clusters or aggregates of bacteria in association with abundant polymers of neutrophil-derived components F-actin and DNA, among other components. These aggregates differ substantially in size and appearance compared to surface-attached in vitro biofilm models classically utilized for studies but are believed to share properties of surface-attached biofilms, including antibiotic resistance. However, little is known about the formation and function of surface-independent modes of biofilm growth, how they might be eradicated, and quorum sensing communication. To address these issues, we developed a novel in vitro model of P. aeruginosa aggregates incorporating human neutrophil-derived products. Aggregates grown in vitro and those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. The lasA quorum sensing gene was differentially expressed in the presence of neutrophil products. Importantly, aggregates formed in the presence of neutrophils acquired resistance to tobramycin, which was lost when the aggregates were dispersed with DNase, and antagonism of tobramycin and azithromycin was observed. This novel yet simple in vitro system advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies against P. aeruginosa.

Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota.

RATIONALE: The airways of people with cystic fibrosis (CF) are chronically infected with a variety of bacterial species. Although routine culture methods are usually used to diagnose these infections, culture-independent, DNA-based methods have identified many bacterial species in CF respiratory secretions that are not routinely cultured. Many prior culture-independent studies focused either on microbiota in explanted CF lungs, reflecting end-stage disease, or those in oropharyngeal swabs, which likely sample areas in addition to the lower airways. Therefore, it was unknown whether the lower airways of children with CF, well before end-stage but with symptomatic lung disease, truly contained diverse microbiota. OBJECTIVES: To define the microbiota in the diseased lung tissue of a child who underwent lobectomy for severe, localized CF lung disease. METHODS: After pathologic examination verified that this child's lung tissue reflected CF lung disease, we used bacterial ribosomal RNA gene pyrosequencing and computational phylogenetic analysis to identify the microbiota in serial sections of the tissue. MEASUREMENTS AND MAIN RESULTS: This analysis identified diverse, and anatomically heterogeneous, bacterial populations in the lung tissue that contained both culturable and nonculturable species, including abundant Haemophilus, Ralstonia, and Propionibacterium species. Routine clinical cultures identified only Staphylococcus aureus, which represented only a small fraction of the microbiota found by sequencing. Microbiota analysis of an intraoperative oropharyngeal swab identified predominantly Streptococcus species. The oropharyngeal findings therefore represented the lung tissue microbiota poorly, in agreement with findings from earlier studies of oropharyngeal swabs in end-stage disease. CONCLUSIONS: These results support the concept that diverse and spatially heterogeneous microbiota, not necessarily dominated by "traditional CF pathogens," are present in the airways of young, symptomatic children with early CF lung disease.

Pseudomonas aeruginosa in vitro phenotypes distinguish cystic fibrosis infection stages and outcomes.

RATIONALE: Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease. OBJECTIVES: To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes. METHODS: A total of 649 children with CF and newly identified P. aeruginosa were followed for a median 5.4 years during which a total of 2,594 P. aeruginosa isolates were collected. Twenty-six in vitro bacterial phenotypes were assessed among the isolates, including measures of motility, exoproduct production, colony morphology, growth, and metabolism. MEASUREMENTS AND MAIN RESULTS: P. aeruginosa phenotypes present at the time of culture were associated with both stage of infection (new onset, intermittent, or chronic) and the primary clinical outcome, occurrence of a pulmonary exacerbation (PE) in the subsequent 2 years. Two in vitro P. aeruginosa phenotypes best distinguished infection stages: pyoverdine production (31% of new-onset cultures, 48% of intermittent, 69% of chronic) and reduced protease production (31%, 39%, and 65%, respectively). The best P. aeruginosa phenotypic predictors of subsequent occurrence of a PE were mucoidy (odds ratio, 1.75; 95% confidence interval, 1.19-2.57) and reduced twitching motility (odds ratio, 1.43; 95% confidence interval, 1.11-1.84). CONCLUSIONS: In this large epidemiologic study of CF P. aeruginosa adaptation, P. aeruginosa isolates exhibited two in vitro phenotypes that best distinguished early and later infection stages. Among the many phenotypes tested, mucoidy and reduced twitching best predicted subsequent PE. These phenotypes indicate potentially useful prognostic markers of transition to chronic infection and advancing lung disease.

Pseudomonas aeruginosa phenotypes associated with eradication failure in children with cystic fibrosis.

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.

Culture-based diagnostic microbiology in cystic fibrosis: can we simplify the complexity?

Cystic fibrosis (CF) diagnostic microbiology has evolved from a focus on Staphylococcus aureus as primary pathogen to identification of the contribution of Pseudomonas aeruginosa and other non-fermenting gram negatives; studies of the lung microbiome have added new complexity. This review summarizes state-of-the art culture methods and makes recommendations for addition of non-culture based methods in the diagnostic laboratory. Plating on selective media is recommended, with organism identification by matrix assisted laser desorption-time of flight mass spectroscopy and real-time polymerase chain reaction (PCR) supplanting both biochemical identification and other less accurate and more time-consuming molecular methods. Conventional antibiotic susceptibility testing, possibly at less frequent intervals, remains the standard but more CF-relevant methods may arise in the future. There is a role for direct identification of organisms in clinical samples using quantitative real-time PCR, next generation sequencing, and metagenomic studies for the re-examination of samples that do not yield traditional CF pathogens.

Pulmonary exacerbations in CF patients with early lung disease.

BACKGROUND: Current definitions of pulmonary exacerbation (PE) in cystic fibrosis are based on studies in participants with significant lung disease and may not reflect the spectrum of findings observed in younger patients with early lung disease. METHODS: We used data from a recent trial assessing the efficacy of azithromycin in children to study signs and symptoms associated with PEs and related changes in lung function and weight. RESULTS: While increased cough was present in all PEs, acute weight loss and reduction in oxygen saturation were not observed. Changes in lung function did not differ between subjects who did experience a PE and those who were exacerbation-free. CONCLUSIONS: Cough was the predominant symptom in CF patients with early lung disease experiencing a PE. There was no significant difference in mean 6-month change in lung function or weight among subjects with one or more exacerbations and those without an exacerbation.

Inhaled aztreonam for chronic Burkholderia infection in cystic fibrosis: a placebo-controlled trial.

BACKGROUND: Individuals with Burkholderia spp. infection have historically been excluded from efficacy trials of inhaled antibiotics, including aztreonam for inhalation solution (AZLI). METHODS: A double-blind, placebo-controlled, 24-week trial of continuous AZLI/placebo treatment was undertaken in individuals with cystic fibrosis (CF) and chronic Burkholderia spp. infection. All subjects also received usual medical care (determined by their physicians). Additional antibiotic use was not restricted. RESULTS: Baseline FEV1% predicted values ranged from 15.8% to 114.6%. No significant treatment differences (AZLI vs. placebo) were observed at week 24 for any endpoints, including FEV1% predicted, number of respiratory exacerbations requiring systemic/inhaled antibiotics, or hospitalizations. Continuous AZLI administration was well tolerated. Burkholderia spp. susceptibility to antibiotics commonly used in CF therapy showed little change. CONCLUSIONS: 24-weeks of continuous AZLI treatment did not significantly improve lung function in CF subjects with chronic Burkholderia spp. infection. Non-study antibiotic use may have confounded any potential AZLI effects.

Developing an international Pseudomonas aeruginosa reference panel.

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is associated with diverse bacteria chronically infecting the airways. Slow-growing, antibiotic-resistant mutants of Staphylococcus aureus known as small-colony variants (SCVs) have been isolated from respiratory secretions from European adults and children with CF lung disease using specific but infrequently used culture techniques. Staphylococcus aureus SCVs can be selected either by exposure to specific antibiotics or by growth with another CF pathogen, Pseudomonas aeruginosa. We sought to determine the prevalence, clinical significance, and likely mechanisms of selection of S. aureus SCVs among a US cohort of children with CF. METHODS: We performed a 2-year study of 100 children with CF using culture techniques sensitive for S. aureus SCVs, and evaluated associations with clinical characteristics using multivariable regression models. RESULTS: Staphylococcus aureus SCV infection was detected among 24% of participants and was significantly associated with a greater drop in lung function during the study (P = .007, adjusted for age and lung function at enrollment). This association persisted after adjusting for infection with other known CF pathogens, including P. aeruginosa and methicillin-resistant S. aureus. Evidence indicated that S. aureus SCVs were likely selected in vivo by treatment with the antibiotic trimethoprim-sulfamethoxazole and possibly by coinfection with P. aeruginosa. CONCLUSIONS: Infection with SCV S. aureus was independently associated with worse CF respiratory outcomes in this pediatric cohort. As many clinical microbiology laboratories do not specifically detect S. aureus SCVs, validation and extension of these findings would require widespread changes in the usual laboratory and clinical approaches to these bacteria.

Sputum tobramycin concentrations in cystic fibrosis patients with repeated administration of inhaled tobramycin.

BACKGROUND: Dosing of tobramycin solution for inhalation (TSI) in cystic fibrosis (CF) patients was based on single-dose pharmacokinetic studies. This investigation was prompted by evidence of possible antibiotic accumulation in respiratory secretions with repeated dosing. The objectives were to evaluate whether tobramycin accumulates in respiratory secretions with repeated inhalation, compare total and biologically active tobramycin concentrations in CF sputum, and evaluate sputum induction for obtaining secretions for drug concentration assay. METHODS: Individuals with CF ≥10 years of age were enrolled at the beginning of a course of TSI, 300 mg twice daily for 28 days. Two study visits were conducted, 1-2 days and 24-28 days after initiation of TSI treatment. Induced sputum and expectorated sputum samples were collected for measurement of trough and peak tobramycin concentrations at each visit. Total tobramycin concentrations were measured by high-pressure liquid chromatography and bioactive concentrations by bioassay. RESULTS: Twenty participants completed the study. Trough concentrations were similar at visits 1 and 2, as were peak concentrations. Trough bioactive and total tobramycin concentrations were similar (mean ratio 1.2, 95% CI 0.56, 1.87), but peak bioactive concentrations were significantly lower than peak total concentrations (mean ratio 0.33, 95% CI 0.23, 0.44). Sputum induction was well tolerated. CONCLUSIONS: No evidence of significant drug accumulation in respiratory secretions with repeat dosing of TSI was seen. Peak bioactive concentrations, although lower than peak total concentrations, were still generally well within the bactericidal range. Sputum induction as a method for determining airway drug concentrations appears safe and feasible.

Pseudomonas aeruginosa isolates of distinct sub-genotypes exhibit similar potential of antimicrobial resistance by drugs exposure.

Pseudomonas aeruginosa, a wide-spread opportunistic pathogen, often complicates clinical treatments due to its resistance to a large variety of antimicrobials, especially in immune compromised patients, occasionally leading to death. However, the resistance to antimicrobials varies greatly among the P. aeruginosa isolates, which raises a question on whether some sub-lineages of P. aeruginosa might have greater potential to develop antimicrobial resistance than others. To explore this question, we divided 160 P. aeruginosa isolates collected from cities of USA and China into distinct genotypes using I-CeuI, a special endonuclease that had previously been proven to reveal phylogenetic relationships among bacteria reliably due to the highly conserved 26-bp recognition sequence. We resolved 10 genotypes by I-CeuI analysis and further divided them into 82 sub-genotypes by endonuclease cleavage with SpeI. Eight of the 10 genotypes contained both multi-drug resistant (MDR) and less resistant isolates based on comparisons of their antimicrobial resistance profiles (ARPs). When the less resistant or susceptible isolates from different genotypes were exposed to eight individual antimicrobials, they showed similar potential to become resistant with minor exceptions. This is to our knowledge the first report to examine correlations between phylogenetic sub-lineages of P. aeruginosa and their potential to become resistant to antimicrobials. This study further alerts the importance and urgency of antimicrobial abuse control.

Pseudomonas aeruginosa syntrophy in chronically colonized airways of cystic fibrosis patients.

Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients undergo remarkable phenotypic divergence over time, including loss of pigmentation, hemolysis, motility, and quorum sensing and emergence of antibiotic hypersusceptibility and/or auxotrophism. With prolonged antibiotic treatment and steady decline in lung function in chronically infected patients, the divergent characteristics associated with CF isolates have traditionally been regarded as "adapted/unusual virulence," despite the degenerative nature of these adaptations. We examined the phenotypic and genotypic diversity in clonally related isogenic strains of P. aeruginosa from individual CF patients. Our observations support a novel model of intra-airway pseudomonal syntrophy and accompanying loss of virulence. A 2007 calendar year collection of CF P. aeruginosa isolates (n = 525) from 103 CF patients yielded in vitro MICs of sulfamethoxazole-trimethoprim (SMX-TMP, which typically has no activity against P. aeruginosa) ranging from 0.02 to >32 µg/ml (median, 1.5). Coisolation of clonally related SMX-TMP-susceptible and -resistant P. aeruginosa strains from the same host was common (57%), as were isogenic coisolates with mutations in efflux gene determinants (mexR, mexAB-oprM, and mexZ) and genes governing DNA mismatch repair (mutL and mutS). In this cohort, complete in vitro growth complementation between auxotrophic and prototrophic P. aeruginosa isogenic strains was evident and concurrent with the coding sequence mosaicism in resistance determinants. These observations suggest that syntrophic clonal strains evolve in situ in an organized colonial structure. We propose that P. aeruginosa adopts a multicellular lifestyle in CF patients due to host selection of an energetically favorable, less-virulent microbe restricted within and symbiotic with the airway over the host's lifetime.