Activity of Cysteamine against the Cystic Fibrosis Pathogen Burkholderia cepacia Complex

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

similar studies (18)(19)(20). Inocula were prepared using a McFarland standard equivalent of 5 ϫ 10 5 CFU/ml from growing cultures (according to CLSI document M07-A9 [15]) into 100 l of cation-adjusted Mueller-Hinton broth containing appropriate concentrations of test antibiotic. Cultures were incubated statically for 48 h in a humidified atmosphere at 37°C to establish biofilms. Culture medium containing planktonic bacteria was carefully removed and discarded, and the plates were washed gently three times with 150 l of sterile phosphate-buffered saline (PBS) prior to air drying for 1 h. The remaining attached bacteria in wells were then stained with 200 l of 1% crystal violet solution for 2 min, prior to 3 further washes with PBS and solubilization with 200 l of ethanol. Plates were then read at 595 nm.
Assessment of cysteamine-mediated biofilm prevention in a shearflow environment was performed using a BioFlux 200 automated microfluidic system (Fluxion Biosciences, CA). Inocula were prepared as described above (CLSI document M07-A9 [15]) from growing cultures and seeded into prewarmed cation-adjusted Mueller-Hinton broth with or without the sub-MIC 100 concentration of 128 g/ml of cysteamine. A 48-well Bioflux plate was primed for 1 min with prewarmed cation-adjusted Mueller-Hinton broth prior to addition of treated and untreated cultures. Medium was passed through capillaries at 37 l/h (0.4 dyne) for 20 h, and images were captured using an Axiovert 40CFL microscope (Carl Zeiss, United Kingdom) and camera and Bioflux 200 software.

RESULTS
MIC assays with combinations of cysteamine and the recommended CF antibiotics were performed to assess the utility of cysteamine in vitro as an adjunct antibiotic therapy against BCC. A panel of BCC clinical isolates and type strains was tested to deter-mine genomovar-specific effects and any differences in cysteamine-mediated effects between type strains and clinical isolates associated with colonization in two major United Kingdom patient cohorts. The 36 strains tested were resistant in vitro to at least one antibiotic selected from tobramycin, ciprofloxacin, trimethoprim-sulfamethoxazole, and ceftazidime (25 out of 36). The majority of clinical strains of CF origin were found to be resistant to tobramycin; perhaps this is not surprising considering the status of tobramycin as a mainstay, long-term antibiotic intervention in CF. Cysteamine potentiated the activity of tobramycin against 33 of 36 BCC isolates and type strains tested and reversed resistance/insensitivity in 17 of those strains (Table 1), all 17 being CF isolates. Cysteamine also potentiated the activity of ciprofloxacin against 21 of 36 BCC isolates tested and reversed resistance/insensitivity in 10 strains that were not sensitive to ciprofloxacin (Table  2). Only two isolates (B. dolosa DSMZ 16088 and a clinical isolate of B. multivorans CFSYN 945) remained resistant to ciprofloxacin in the presence of cysteamine.
In addition to this impact on widely used antibiotics, the ability of cysteamine to enhance the activity of ceftazidime and trimethoprim-sulfamethoxazole, antibiotics used specifically to treat BCC, was also investigated (Tables 3 and 4). Interestingly, and pointing to an antibiotic class-specific effect, cysteamine had no major impact on ceftazidime susceptibility of the BCC strains tested in this system. In contrast, cysteamine potentiated the activity of trimethoprim-sulfamethoxazole against the majority (22 out of 36) of BCC isolates and type strains studied.
Planktonic cells were employed in our initial cysteamine adjunct assays, whereas in vivo, BCC colonizes the CF airway in biofilm form (21,22). We have already described the antibiofilm properties of cysteamine against Pseudomonas (10)(11)(12). In order to confirm any activity of cysteamine specifically against BCC biofilm structures in vitro, we assessed its ability to prevent BCC biofilm formation using the Bioflux microfluidic system, and we used the crystal violet method for assessing biomass in 96-well microtiter plates. In both systems, the methodology was adapted to keep conditions as close as possible to CLSI standards for MIC testing rather than to favor the growth of biofilms. For example, the medium used was cationadjusted Mueller-Hinton broth as opposed to other choices, such as tryptic soy broth, which would favor biofilm formation in BCC (23). This was to clearly demonstrate inhibition of attachment at sub-MIC 100 s. Crystal violet assessment demonstrated inhibition of bacterial attachment at 48 h in the presence of concentrations of cysteamine subinhibitory for planktonic growth (Fig. 1). Inhibition of biofilm formation was dose dependent, increasing with concentrations of cysteamine approaching the MIC, with significant inhibition of both strains of B. cenocepacia tested at 128 g/ml. The antibiofilm activity of cysteamine against BCC was confirmed in real time in a dynamic-flow microfluidics system. The effect of a subinhibitory concentration of cysteamine was assessed on biofilm formation in the BioFlux microfluidic system on B. cenocepacia clinical strain CFSYN 1112. Cysteamine prevented biofilm formation in cells in channels that were exposed to cysteamine over 20 h compared to samples in wells not exposed to cysteamine (see Movie S1 in the supplemental material). Viable planktonic cells from the outlet well were cultured at the end of the experiment, confirming an effect on attachment and not bacterial viability at the test concentration.

DISCUSSION
The findings of our current study point to the potential of cysteamine as a means to resolve or prevent BCC colonization through a simple and sustainable modification to the current standard of care in CF. Tobramycin and ciprofloxacin are mainstays of the CF antibiotic regimen, and resistance to these antibiotics in CF BCC strains is common and inherent in some strains (24) and is readily selected for (25); indeed, we demonstrate that all but one of the strains (NCTC 13008) tested in this study which had been isolated from a patient with CF were resistant to tobramycin treatment. Cysteamine was able to reverse the tobramycin and cipro- floxacin resistance phenotype and improve sensitivity to co-trimoxazole treatment (Tables 1 to 3). Therefore, cysteamine has the potential to extend the target spectrum of these antibiotics to include BCC. This is timely as regards its potential use as an adjunct with tobramycin considering the recent increased interest in the reapplication of inhaled or high-dose tobramycin against BCC (26,27). Furthermore, the activity of antibiotics specifically deployed against BCC, such as trimethoprim-sulfamethoxazole, can also be further potentiated by cysteamine. Interestingly, ceftazidime activity was not altered by cotreatment with cysteamine, which suggests an antibiotic class-specific effect, at least within the in vitro systems employed in this study to assess antimicrobial activity.
The slow-growing, biofilm-forming characteristic of BCC contributes to the recalcitrance of this organism to existing antibiotic chemotherapy. In this study, we followed our previous work on the interactions of cysteamine with biofilm (10) in addition to assessing any direct antimicrobial activity. Cysteamine inhibited bacterial attachment at concentrations below the MIC 100 for each strain tested, with significant inhibition of B. cenocepacia type strain DSMZ 16553 and clinical isolate CFSYN 1112. Interestingly, although we previously demonstrated that combinations of tobramycin and cysteamine were more effective in biofilm prevention and eradication for Pseudomonas aeruginosa, the addition of antibiotics did not enhance the antibiofilm activity of cysteamine against BCC (data not shown). Cysteamine was not able to disrupt existing biofilms in the slower-growing BCC strains over 48 h over the same range of concentrations of the antibiotics as tested in this in vitro system. This may indicate that cysteamine adjunct maintenance therapy may be better at preventing the establishment of BCC colonization in CF than at removing existing biofilms in chronically infected patients; however, the enhancement of antimicrobial activity may prove to be the more important feature of this compound in this situation. Further research to determine optimum antibiotic combinations and concentrations to eradicate established BCC biofilms may yet prove efficacious. We purposely did not use an exhaustive panel of BCC strains for this study. We instead employed a focused set of clinically relevant CF isolates from two of the United Kingdom's specialist CF centers (8 isolates from Glasgow and 16 from Aberdeen) and an additional 12 type strains in order to cover all known BCC genomovars, regardless of clinical relevance.
Cysteamine is in late-stage clinical trials for the treatment of cystic fibrosis and is being developed in oral and inhaled forms for acute exacerbations and chronic longer-term maintenance (10)(11)(12). An oral form of cysteamine was investigated in an open-label clinical study (28) in the United Kingdom in which tolerability, absorption, pK, and early evidence of efficacy were assessed in adult CF patients with stable disease. A global twopart registration study for oral cysteamine in acute exacerbations is now being initiated (EudraCT no. 2015-0004986-99) for which endpoints will include the reduction in sputum microbial burden over and above that achieved with standard of care therapy (SOCT) exacerbation interventions.
Thus far, cysteamine appears to be a promising candidate treatment for CF, but how its interactions with all components of the complex CF microbiome contribute to its clinical effects is yet to be determined. We have already have demonstrated the utility of cysteamine against other, more common CF pathogens that are known to drive acute infectious exacerbations (Pseudomonas in particular) (29,30). We believe that this study is important in confirming the efficacy of cysteamine against the more insidious BCC and its colonization of the CF airway, which may be eradicated and perhaps prevented by long-term use of an adjunct to SOCT such as cysteamine, which is able to potentiate the effects of existing antibiotics and "switch" BCC to becoming sensitive and also prevent this organism from forming biofilms. Not all BCC isolates tested in this study responded to cotreatment. As well as any strain-specific nuances in cysteamine response, the antibiotic class-specific differences in responses to cysteamine coexposure we have underpinned for BCC (and other organisms in our previous work) are the subject of further study.