Michael D. Parkins

A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. A modest number of bacterial pathogens have been correlated with pulmonary function decline; however, microbiological and molecular evidence suggests that CF airway infection is polymicrobial. To obtain a more complete assessment of the microbial community composition and dynamics, we undertook a longitudinal study by using culture-independent and microbiological approaches. In the process, we demonstrated that within complex and dynamic communities, the Streptococcus milleri group (SMG) can establish chronic pulmonary infections and at the onset of 39% of acute pulmonary exacerbations, SMG is the numerically dominant pathogen. We report the comprehensive polymicrobial community dynamics of a CF lung infection in a clinically relevant context. If a given organism, such as Pseudomonas aeruginosa, becomes resistant to antibiotic therapy, an alternative treatment avenue may mediate the desired clinical response by effectively managing the composition of the microbial community.

Multidrug Efflux Pumps: Expression Patterns and Contribution to Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

ABSTRACT Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance ofP. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA andmexC in the developing biofilm. Remarkably, expression ofmexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM andmexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation.

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two‐component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10‐fold reduction in biofilm formation capacity resulted relative to wild‐type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA−. Providing gacA in trans on the multicopy vector pUCP‐gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA− revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA− twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar‐mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA− biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.

Discerning the Complexity of Community Interactions Using a Drosophila Model of Polymicrobial Infections

A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe–microbe interactions and polymicrobe–host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe–microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured.

Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.

Population‐Based Epidemiological Study of Infections Caused by Carbapenem‐Resistant Pseudomonas aeruginosa in the Calgary Health Region: Importance of Metallo‐β‐Lactamase (MBL)–Producing Strains

BACKGROUND A study was conducted in the Calgary Health Region between May 2002 and April 2004 to define the population-based epidemiological characteristics of infections caused by imipenem-resistant Pseudomonas aeruginosa and to explore the clinical outcomes due to metallo- beta -lactamase (MBL)-producing and non-MBL-producing strains. METHODS Detailed clinical information was obtained by chart review, and phenotypic and molecular characterizations were performed using the MBL E-test, polymerase chain reaction with sequencing, and pulsed-field gel electrophoresis. RESULTS A total of 228 patients with infections caused by imipenem-resistant P. aeruginosa were identified (annual incidence, 10.5 cases/100,000 population), with the highest incidence rate in those >or=75 years old. MBL-producing strains (98/228) were associated with higher rates of multidrug resistance and bacteremia. Ninety MBL-producing strains also produced VIM-2, 4 produced IMP-7, and 4 were unclassified. A cluster of VIM-2-producing strains was responsible for a nosocomial outbreak during 2003. The case-fatality rate was significantly higher for infections caused by MBL-producing strains than for those caused by non-MBL-producing strains (25% vs. 13%; relative risk, 1.98 [95% confidence interval, 1.00-3.90]; P=.05). CONCLUSION MBL-producing P. aeruginosa strains were associated with a higher case-fatality rate and invasive disease. Our study highlights the potential importance of molecular laboratory techniques in infection control and patient care.

Emerging bacterial pathogens and changing concepts of bacterial pathogenesis in cystic fibrosis

Chronic suppurative lower airway infection is a hallmark feature of cystic fibrosis (CF). Decades of experience in clinical microbiology have enabled the development of improved technologies and approaches for the cultivation and identification of microorganisms from sputum. It is increasingly apparent that the microbial constituents of the lower airways in CF exist in a dynamic state. Indeed, while changes in prevalence of various pathogens occur through ageing, differences exist in successive cohorts of patients and between clinics, regions and countries. Classical pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex and Staphylococcus aureus are increasingly being supplemented with new and emerging organisms rarely observed in other areas of medicine. Moreover, it is now recognized that common oropharyngeal organisms, previously presumed to be benign colonizers may contribute to disease progression. As infection remains the leading cause of morbidity and mortality in CF, an understanding of the epidemiology, risk factors for acquisition and natural history of infection including interactions between colonizing bacteria is required. Unified approaches to the study and determination of pathogen status are similarly needed. Furthermore, experienced and evidence-based treatment data is necessary to optimize outcomes for individuals with CF.

The Streptococcus milleri group—An unrecognized cause of disease in cystic fibrosis: A case series and literature review

The “Streptococcus milleri” group (SMG) is increasingly recognized for their role in pyogenic infections including empyema and solid organ abscesses. However, SMG disease has rarely been identified in cystic fibrosis (CF). Inherent difficulties in both growing the organisms and distinguishing SMG from less virulent oropharyngeal viridans streptococci may have led to a decreased recognition of this as a CF pathogen. We report on six cases of SMG‐related infection over a 4‐year time‐frame occurring within an adult CF clinic in Canada, and a further four cases identified through a literature review. SMG manifested disease as bronchopulmonary exacerbations in 7 of 10 patients, and 4 of 10 patients had extra‐pulmonary dissemination of SMG infection. Noticeably, pulmonary exacerbations were frequently associated with atypically malodorous sputum. Furthermore, patients clinically responded to anti‐microbial therapies with no anti‐Pseudomonal activity. There was a consistent correlation of SMG disease and co‐colonization with P. aeruginosa leading to speculation of polymicrobial interactions resulting in enhanced virulence. SMG deserves considerable attention as a potential pathogen within the airways of patients with CF. Pediatr Pulmonol. 2008; 43:490–497.

Incidence and risk factors for pulmonary exacerbation treatment failures in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa.

BACKGROUND Pulmonary exacerbations (PEx) are responsible for much of the morbidity and mortality associated with cystic fibrosis (CF). However, there is a paucity of data on outcomes in CF PEx and factors influencing outcomes. METHODS We reviewed all PEx in patients infected with Pseudomonas aeruginosa treated with parenteral antibiotics over 4 years at our center. Treatment failures were categorized a priori as those PEx requiring antibiotic regimen change, prolongation of therapy > 20 days because of failure to respond, an early recurrent event within < 45 days, or failure to recover lung function to > 90% of baseline FEV(1). RESULTS A total of 101 patients were followed for 452 PEx. Treatment failures were observed in 125 (28%) of PEx; antibiotic regimen change was observed in 27 (6%), prolongation of therapy in 29 (6%), early recurrent events in 63 (14%), and failure to recover lung function to > 90% of baseline FEV(1) in 66 (15%). Demographic factors associated with one or more treatment failures per year included advanced airways disease, use of enteric feeds, CF-related diabetes, and CF liver disease but did not include female sex or F508del homozygosity. Increased treatment failure risk was associated with lower admission FEV(1) and increased markers of inflammation. At therapeutic completion, increased inflammatory markers correlated with treatment failure. Failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility (zero, 28/65 [43%]; one, 38/140 [27%]; two, 59/245 [24%]; three, 0/2 [0%]; P = .02). CONCLUSIONS One-fourth of PEx fail to respond adequately to initial management. Patient demographic and episode-specific clinical information can be used to identify individuals at increased risk of initial management failure.