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Many Pseudomonas aeruginosa isolates from the airways of patients with cystic fibrosis (CF) are sensitive to antibiotics in susceptibility testing, but eradication of the infection is difficult. The main reason is the biofilm formation in the airways of patients with CF. The pharmacokinetics (PKs) and pharmacodynamics (PDs) of antimicrobials can reliably be used to predict whether antimicrobial regimens will achieve the maximum bactericidal effect against infections. Unfortunately, however, most PK/PD studies of antimicrobials have been done on planktonic cells and very few PK/PD studies have been done on biofilms, partly due to the lack of suitable models in vivo. In the present study, a biofilm lung infection model was developed to provide an objective and quantitative evaluation of the PK/PD profile of antimicrobials. Killing curves were set up to detect the antimicrobial kinetics on planktonic and biofilm P. aeruginosa cells in vivo. Colistin showed concentration-dependent killing, while imipenem showed time-dependent killing on both planktonic and biofilm P. aeruginosa cells in vivo. The parameter best correlated to the elimination of bacteria in lung by colistin was the area under the curve (AUC) versus MIC (AUC/MIC) for planktonic cells or the AUC versus minimal biofilm inhibitory concentration (MBIC; AUC/MBIC) for biofilm cells. The best-correlated parameter for imipenem was the time that the drug concentration was above the MIC for planktonic cells (TMIC) or time that the drug concentration was above the MBIC (TMBIC) for biofilm cells. However, the AUC/MIC of imipenem showed a better correlation with the efficacy of imipenem for biofilm infections (R2 = 0.89) than planktonic cell infections (R2 = 0.38). The postantibiotic effect (PAE) of colistin and imipenem was shorter in biofilm infections than planktonic cell infections in this model.

Recent studies have determined that Pseudomonas aeruginosa can live in a biofilm mode within hypoxic mucus in the airways of patients with cystic fibrosis (CF). P. aeruginosa grown under anaerobic and biofilm conditions may better approximate in vivo growth conditions in the CF airways, and combination antibiotic susceptibility testing of anaerobically and biofilm-grown isolates may be more relevant than traditional susceptibility testing under planktonic aerobic conditions. We tested 16 multidrug-resistant isolates of P. aeruginosa derived from CF patients using multiple combination bactericidal testing to compare the efficacies of double and triple antibiotic combinations against the isolates grown under traditional aerobic planktonic conditions, in planktonic anaerobic conditions, and in biofilm mode. Both anaerobically grown and biofilm-grown bacteria were significantly less susceptible (P < 0.01) to single and combination antibiotics than corresponding aerobic planktonically grown isolates. Furthermore, the antibiotic combinations that were bactericidal under anaerobic conditions were often different from those that were bactericidal against the same organisms grown as biofilms. The most effective combinations under all conditions were colistin (tested at concentrations suitable for nebulization) either alone or in combination with tobramycin (10 μg ml−1), followed by meropenem combined with tobramycin or ciprofloxacin. The findings of this study illustrate that antibiotic sensitivities are dependent on culture conditions and highlight the complexities of choosing appropriate combination therapy for multidrug-resistant P. aeruginosa in the CF lung.

Evidence suggests that Pseudomonas aeruginosa bacteria form biofilms within the airways of adults with cystic fibrosis (CF). The objective of this study was to determine whether clinical isolates of P. aeruginosa recovered from adults with CF have similar susceptibilities to individual antibiotics and to antibiotic combinations when grown as adherent monolayers or as biofilms compared to when they are grown using planktonic methods. Twelve multiresistant P. aeruginosa isolates, one mucoid and one nonmucoid from each of six CF patients, were grown conventionally under planktonic conditions, as adherent bacterial monolayers, and as biofilms. Each bacterial isolate remained genotypically identical despite being cultured under planktonic, adherent, or biofilm growth conditions. Isolates grown as adherent monolayers and as biofilms were less susceptible to bactericidal killing by individual antibiotics compared to those grown planktonically. More importantly, biofilm-grown bacteria, but not adherent monolayer-grown bacteria, were significantly less susceptible to two- and three-drug combinations of antibiotics than were planktonically grown bacteria (P = 0.005). We conclude that biofilm-grown bacteria derived from patients with CF show decreased susceptibility to the bactericidal effects of antibiotic combinations than do adherent and planktonically grown bacteria.

Multiresistant Pseudomonas aeruginosa isolates can chronically infect patients with cystic fibrosis. Acute infective exacerbations are treated with combinations of two antipseudomonal antibiotics. Patients may respond clinically even if the bacteria are resistant, possibly due to antimicrobial synergy. The challenge for testing for synergy in vitro is that there is no standardized method, and the antibiotic susceptibility in a population of P. aeruginosa isolates in a single sputum sample can vary. We therefore compared (i) antibiotic combinations with different examples of resistant bacteria from the same sputum sample and (ii) the results of synergy testing by different methods. Antibiotic synergy was tested by using resistant P. aeruginosa isolates recovered from sputum samples taken just before the start of treatment for an acute infective exacerbation. Several examples of each morphotype of P. aeruginosa were tested by cidal checkerboard, time-kill curve, and multiple-combination bactericidal testing. The isolates were typed by pulsed-field gel electrophoresis (PFGE). The results were compared with the clinical and microbiological responses to 14 days of antibiotic treatment. Forty-four resistant isolates from nine patients were tested. Some P. aeruginosa isolates with the same morphotype and PFGE pulsotype had different results by synergy testing. There was a poor correlation between the results of the different methods of synergy testing, and no one method would have predicted the response to treatment in all patients. The in vitro effects of antibiotic combinations against different isolates from the same sputum sample can vary, and the results depend on the methodology used. The role of combination testing for the treatment of antibiotic-resistant P. aeruginosa in acute exacerbations of chronic infection in patients with cystic fibrosis needs to be reviewed.

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

Author Summary

Pseudomonas aeruginosa causes serious infections in people with compromised immune systems. Individuals with Cystic Fibrosis and hospital patients are particularly vulnerable to P. aeruginosa infections. This bacterium does not respond to many antibiotics, making these infections difficult to treat. P. aeruginosa can grow as free-floating planktonic cells or as microcolonies known as biofilms. The ability of P. aeruginosa to form biofilms is thought to contribute to their ability to cause chronic infections. The aim of this research was to develop a simple biofilm model of infection using the fruit fly (Drosophila melanogaster). The immune system of the fruit fly has similarities with the vertebrate innate immune system. Understanding how P. aeruginosa causes infections in Drosophila will aid in understanding virulence mechanisms in mammals. In this study we show that feeding P. aeruginosa to Drosophila results in a biofilm infection and biofilm infections induced expression of antimicrobial peptide immune response genes in the fly. Using fly survival as a measure of virulence we showed that biofilm infections were less virulent than non-biofilm infections. These results provide novel insight into host-pathogens interactions during P. aeruginosa infection.

Pseudomonas aeruginosa is the predominant cause of chronic airway infection in cystic fibrosis (CF). CF airway isolates are often tested for antibiotic susceptibility but are rarely eradicated by the antibiotics identified as potentially effective. The growth state of P. aeruginosa in CF airways is probably different from that exhibited under conventional susceptibility testing conditions and may represent a bacterial biofilm. Biofilm susceptibility testing methods were adapted to create an assay for implementation in a clinical microbiology laboratory. This assay gave reproducible results when examined in 300 paired determinations with 12 antimicrobial agents, with a serious error rate of 5.7%. The biofilm assay was used retrospectively to test these 12 agents against 94 isolates from 41 CF patients. The biofilm inhibitory concentrations (BICs) were much higher than the corresponding conventionally determined MICs for the β-lactam antibiotics (median values: aztreonam, >128 μg/ml versus 4 μg/ml; ceftazidime, 128 μg/ml versus 2 μg/ml; piperacillin-tazobactam, 256 μg/ml versus 4 μg/ml; and ticarcillin-clavulanate, 512 μg/ml versus 16 μg/ml, respectively) and doxycycline (>64 μg/ml versus 16 μg/ml); and similar for meropenem (4 μg/ml versus ≤ 1 μg/ml), ciprofloxacin (0.5 μg/ml versus 1 μg/ml), and the aminoglycosides amikacin (32 μg/ml versus 16 μg/ml), gentamicin (16 μg/ml versus 8 μg/ml), and tobramycin (4 μg/ml versus 2 μg/ml). The median BIC for azithromycin was 2 μg/ml, whereas isolates were uniformly resistant when tested by standard methods. This demonstrates the feasibility of adapting biofilm susceptibility methods to the clinical microbiology laboratory and opens the way to examining whether biofilm testing might be used to select more effective antibiotic combinations for CF airway infections than methods in current use.

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.

Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.

An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.

The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.

During the course of chronic cystic fibrosis (CF) infections, Pseudomonas aeruginosa undergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. Chronic P. aeruginosa infections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginate-overproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The screen is a luminescence-based attachment assay that has been validated with several strains of P. aeruginosa and compared to a well-established but low-throughput crystal violet staining biofilm assay. P. aeruginosa strain PAO1 was selected for use in the screen both because it forms robust biofilms and because genetic information and tools are available for the organism. The attachment-inhibited mutant, strain PAO1 ΔfliC, was used as a screening-positive control. We have also developed and validated a complementary biofilm detachment assay that can be used as an alternative primary screen or secondary screen for the attachment screening-positive compounds. We have determined the potencies of 61 compounds against biofilm attachment and have identified 30 compounds that fall into different structural classes as biofilm attachment inhibitors with 50% effective concentrations of less than 20 μM. These small-molecule inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections.

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two-component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent posttranslational control, specifically governing type III cytotoxin secretion, exists in this species.

Background

Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro.

Results

Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture.

Conclusions

Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.

Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 μg/ml tobramycin was administered with azithromycin at 2 μg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 μg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 μg/ml combined with clarithromycin at 200 μg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections.

ABSTRACT

Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107 to 2 × 107 P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients.

IMPORTANCE

Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

Staphylococcus lugdunensis is an atypically virulent coagulase-negative staphylococcal species associated with acute and destructive infections that often resemble Staphylococcus aureus infections. Several types of infection caused by S. lugdunensis (e.g., native valve endocarditis, prosthetic joint infection, and intravascular catheter infection) are associated with biofilm formation, which may lead to an inability to eradicate the infection due to the intrinsic nature of biofilms to resist high levels of antibiotics. In this study, planktonic MICs and MBCs and biofilm bactericidal concentrations of 10 antistaphylococcal antimicrobial agents were measured for 15 S. lugdunensis isolates collected from patients with endocarditis, medical device infections, or skin and soft tissue infections. Planktonic isolates were susceptible to all agents studied, but biofilms were resistant to high concentrations of most of the drugs. However, moxifloxacin was able to kill 73% of isolates growing in biofilms at ≤0.5 μg/ml. Relative to the effect on cell density, subinhibitory concentrations of nafcillin substantially stimulated biofilm formation of most isolates, whereas tetracycline and linezolid significantly decreased biofilm formation in 93 and 80% of isolates, respectively. An unexpected outcome of MBC testing was the observation that vancomycin was not bactericidal against 93% of S. lugdunensis isolates, suggesting widespread vancomycin tolerance in this species. These data provide insights into the response of S. lugdunensis isolates when challenged with various levels of antimicrobial agents in clinical use.

SUMMARY

Rationale

In cystic fibrosis (CF), conventional antibiotic susceptibility results correlate poorly with clinical outcomes. We hypothesized that biofilm testing would more accurately reflect the susceptibilities of bacteria infecting CF airways.

Methods

A multi-center randomized pilot trial was conducted to assess the efficacy and safety of using biofilm susceptibility testing of Pseudomonas aeruginosa sputum isolates to guide antibiotic regimens for chronic airway infections in clinically stable adolescent and adult CF patients. Thirty-nine participants were randomized to biofilm or conventional treatment groups; 14-day courses of two antibiotics were selected according to an activity-based algorithm using the corresponding susceptibility results.

Results

Of the agents tested, meropenem was most active against biofilm-grown bacteria, and was included in regimens for about half of each study group. For nineteen of 39 randomized participants, randomization to the other study group would not have changed the antibiotic classes of the assigned regimen. Study groups were comparable at baseline, and had similar mean decreases in bacterial density, measured in log10 colony forming units per gram of sputum (biofilm, -2.94 [SD 2.83], versus conventional, -3.27 [SD 3.09]), and mean increases in forced expiratory volume in one second, measured in liters (0.18 [SD 0.20] versus 0.12 [SD 0.22]).

Conclusions

In this pilot study, antibiotic regimens based on biofilm testing did not differ significantly from regimens based on conventional testing in terms of microbiological and clinical responses. The predictive value of biofilm testing may nonetheless warrant evaluation in an adequately powered clinical trial in younger CF patients or those experiencing acute pulmonary exacerbation.

The development of resistance to β-lactam antibiotics is a problem in the treatment of chronic Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. The main resistance mechanism is high-level expression of the chromosomally encoded AmpC β-lactamase of P. aeruginosa cells growing in biofilms. Several genes have been shown to influence the level of ampC expression, but little is known about the regulation of ampC expression in P. aeruginosa biofilms. To study the expression of ampC in P. aeruginosa biofilms, we constructed a reporter that consisted of the fusion of the ampC promoter to gfp(ASV) encoding an unstable version of the green fluorescent protein. In vitro biofilms of P. aeruginosa were exposed to the β-lactam antibiotics imipenem and ceftazidime. Sub-MICs of imipenem significantly induced the monitor system of the biofilm bacteria in the peripheries of the microcolonies, but the centers of the microcolonies remained uninduced. However, the centers of the microcolonies were physiologically active, as shown by experiments with another monitor construction consisting of an arabinose-inducible promoter fused to gfp(ASV). The whole biofilm was induced in the presence of increased imipenem concentrations. Ceftazidime induced the monitor system of the biofilm bacteria as well, but only bacteria in the peripheries of the microcolonies were induced in the presence of even very high concentrations. The experiments illustrate for the first time the dynamic and spatial distributions of β-lactamase induction in P. aeruginosa cells growing in biofilms. Thus, our experiments show that P. aeruginosa cells growing in biofilms constitute a heterogeneous population unit which may create different antibiotic-selective environments for the bacteria in the biofilm.

P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycin-treated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 μg/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is caused by biofilm-growing mucoid strains. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy, and they can be treated by chronic suppressive therapy. New results from one small trial suggest that addition of oral ciprofloxacin to inhaled tobramycin may reduce lung inflammation. Clinical trials with new formulations of old antibiotics for inhalation therapy (aztreonam lysine) against chronic P. aeruginosa infection improved patient-reported outcome, lung function, time to acute exacerbations and sputum density of P. aeruginosa. Other drugs such as quinolones are currently under investigation for inhalation therapy. A trial of the use of anti-Pseudomonas antibiotics for long-term prophylaxis showed no effect in patients who were not already infected. Use of azithromycin to treat CF patients without P. aeruginosa infection did not improve lung function. Here I review the recent advances in the treatment of P. aeruginosa lung infections with a focus on inhalation treatments targeted at prophylaxis and chronic suppressive therapy.

Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.

The lungs of cystic fibrosis (CF) patients are commonly colonized with Pseudomonas aeruginosa biofilms. Chronic endobronchial P. aeruginosa infections are impossible to eradicate with antibiotics, but intensive suppressive antibiotic therapy is essential to maintain the lung function of CF patients. The treatment often includes β-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a β-lactam antibiotic, imipenem, on gene expression in biofilm populations. Many genes showed small but statistically significant differential expression in response to imipenem. We identified 34 genes that were induced or repressed in biofilms exposed to imipenem more than fivefold compared to the levels of induction or repression for the controls. As expected, the most strongly induced gene was ampC, which codes for chromosomal β-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated from chronically colonized lungs of CF patients are nearly always mucoid due to the overproduction of alginate. Exposure to subinhibitory concentrations of imipenem caused structural changes in the biofilm, e.g., an increased biofilm volume. Increased levels of alginate production may be an unintended adverse consequence of imipenem treatment in CF patients.

Biofilms are thought to play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, 20 nonmucoid P. aeruginosa isolates collected during different periods of chronic infection from eight CF patients were assessed with respect to phenotypic changes and in vitro biofilm formation. The physiological alterations were associated with a loss of motility (35% were nonmotile) and with decreased production of virulence factors (pyocyanin, proteases) and quorum-sensing molecules (45% of the isolates were unable to produce 3-O-C12-homoserine lactone quorum-sensing molecules). Compared with wild-type strain PAO1, most P. aeruginosa isolates demonstrated different degrees of reduction of adherence on polystyrene surfaces. The in vitro biofilm formation of isolates was investigated in a hydrodynamic flow system. Confocal laser scanning microscope analysis showed that the biofilm structures of the P. aeruginosa isolates were highly variable in biomass and morphology. Biofilm development of six genotypically identical sequential isolates recovered from a particular patient at different time points of chronic infection (20 years) and after lung transplantation demonstrated significant changes in biofilm architectures. P. aeruginosa biofilm formation followed a trend of decreased adherence with progression of the chronic lung infection. The results suggest that the adherent characteristic of in vitro biofilm development was not essential for the longitudinal survival of nonmucoid P. aeruginosa during chronic lung colonization.