P. aeruginosa adaptation in cystic fibrosis environment challenged by the presence of other bacteria

Abstract

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in the cystic fibrosis transmembrane regulator resulting in thick mucus that difficults proper airflow. CF airways are infected by several opportunistic pathogens but P. aeruginosa is the major responsible for the high mortality rate due to the development of chronic infections. To long persist, P. aeruginosa uses sophisticated mechanisms to achieve full-adaptation mainly triggered by the harsh environmental conditions of CF lungs. CF lungs are colonized by several other bacterial species, as the early colonizers, with which P. aeruginosa have to co-exist. Some interactions between the typical early colonizers of CF lungs, such as S. aureus, S. maltophilia, and P. aeruginosa, have been reported. For instance, it has been proposed that S. aureus may somewhat help P. aeruginosa adaptation to CF lungs inducing the emergence of P. aeruginosa small colony variants (SCV). P. aeruginosa SCV are known to be excellent biofilm formers and typically exhibit resistance against multiple antibiotic classes. But, the current in vitro studies have using models that do not closely mimic the complex CF environment. Therefore, the main objective of this study was to determine the impact of S. maltophilia and S. aureus on P. aeruginosa pathogenicity using a new in vitro model that mimics CF lungs environment. To accomplish this feature, a clinical isolate of S. maltophilia and two P. aeruginosa strains, one antibiotic sensitive and one resistant, were grown in artificial CF sputum medium (ASM) and cultured at different timings to simulate a typical CF lung colonization. S. maltophilia was first cultured for 3 days, and then P. aeruginosa was co-cultured for more 4 days. In the last 4 days the mixed cultures were exposed to aggressive ciprofloxacin treatment. Samples were collected every 24 h to investigate bacterial growth kinetics, ciprofloxacin time killing and phenotypic diversity. The same procedure was carried out to assess the impact of S. aureus (an antibiotic sensitive strain and one resistant) on P. aeruginosa. To gain insights in the biological processes involved in P. aeruginosa adaptation in ASM and response to ciprofloxacin, the proteomic profile of P. aeruginosa strains was studied using a 2-D gel electrophoresis (2-DE). Growth kinetics showed that apparently P. aeruginosa outcompeted S. maltophilia in ASM. Currently, there is no solid media able to effectively distinguish P. aeruginosa from S. maltophilia and without this differentiation it was not possible to infer if the outcompetition occurred indeed in ASM or in solid medium. Therefore, other methods, such as fluorescent differentiation or molecular methods, have to be used. Data showed that P. aeruginosa growth kinetics and phenotypic diversity were not affected by the presence of S. aureus. Interestingly, the presence of S. aureus seemed to inhibit the emergence of P. aeruginosa SCV that have emerged when the resistant strain was cultured alone. These results do not substantiate the role of S. aureus on P. aeruginosa persistence pointed out by other studies where it was reported that S. aureus extracellular factors increased P. aeruginosa growth activity and resistance towards antibiotics by the emergence of SCV. It was hypothesized that these discrepancies among results could be caused by the effect of initial P. aeruginosa concentration or by the short period of time for S. aureus adapt to CF environment. The effect of P. aeruginosa concentration revealed no impact on P. aeruginosa adaptation. In contrast, CF-adapted S. aureus seemed to trigger the emergence of P. aeruginosa SCV, but the P. aeruginosa resistant strain was also able to produce SCV when grown alone in ASM. Therefore, both hypothesis were discarded which led to conclude that S. aureus seemed to have no role in P. aeruginosa pathogenicity in CF lungs. It was also concluded that the discrepancy of this study with literature may be explained by the different in vitro conditions used. The majority of the studies used standard laboratory medium that does not fully mimic the chemical and nutritional complex environment found in CF lungs. Moreover, this model used different inoculation timings of bacterial species to better mimic in vivo conditions, issue that was not took into account in the previous studies. 2-DE revealed some differences in protein expression by P. aeruginosa strains that could be involved in important biological processes, such as iron transport, carbon catabolism and lipids biosynthesis.

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in the cystic fibrosis transmembrane regulator resulting in thick mucus that difficults proper airflow. CF airways are infected by several opportunistic pathogens but P. aeruginosa is the major responsible for the high mortality rate due to the development of chronic infections. To long persist, P. aeruginosa uses sophisticated mechanisms to achieve full-adaptation mainly triggered by the harsh environmental conditions of CF lungs. CF lungs are colonized by several other bacterial species, as the early colonizers, with which P. aeruginosa have to co-exist. Some interactions between the typical early colonizers of CF lungs, such as S. aureus, S. maltophilia, and P. aeruginosa, have been reported. For instance, it has been proposed that S. aureus may somewhat help P. aeruginosa adaptation to CF lungs inducing the emergence of P. aeruginosa small colony variants (SCV). P. aeruginosa SCV are known to be excellent biofilm formers and typically exhibit resistance against multiple antibiotic classes. But, the current in vitro studies have using models that do not closely mimic the complex CF environment. Therefore, the main objective of this study was to determine the impact of S. maltophilia and S. aureus on P. aeruginosa pathogenicity using a new in vitro model that mimics CF lungs environment. To accomplish this feature, a clinical isolate of S. maltophilia and two P. aeruginosa strains, one antibiotic sensitive and one resistant, were grown in artificial CF sputum medium (ASM) and cultured at different timings to simulate a typical CF lung colonization. S. maltophilia was first cultured for 3 days, and then P. aeruginosa was co-cultured for more 4 days. In the last 4 days the mixed cultures were exposed to aggressive ciprofloxacin treatment. Samples were collected every 24 h to investigate bacterial growth kinetics, ciprofloxacin time killing and phenotypic diversity. The same procedure was carried out to assess the impact of S. aureus (an antibiotic sensitive strain and one resistant) on P. aeruginosa. To gain insights in the biological processes involved in P. aeruginosa adaptation in ASM and response to ciprofloxacin, the proteomic profile of P. aeruginosa strains was studied using a 2-D gel electrophoresis (2-DE). Growth kinetics showed that apparently P. aeruginosa outcompeted S. maltophilia in ASM. Currently, there is no solid media able to effectively distinguish P. aeruginosa from S. maltophilia and without this differentiation it was not possible to infer if the outcompetition occurred indeed in ASM or in solid medium. Therefore, other methods, such as fluorescent differentiation or molecular methods, have to be used. Data showed that P. aeruginosa growth kinetics and phenotypic diversity were not affected by the presence of S. aureus. Interestingly, the presence of S. aureus seemed to inhibit the emergence of P. aeruginosa SCV that have emerged when the resistant strain was cultured alone. These results do not substantiate the role of S. aureus on P. aeruginosa persistence pointed out by other studies where it was reported that S. aureus extracellular factors increased P. aeruginosa growth activity and resistance towards antibiotics by the emergence of SCV. It was hypothesized that these discrepancies among results could be caused by the effect of initial P. aeruginosa concentration or by the short period of time for S. aureus adapt to CF environment. The effect of P. aeruginosa concentration revealed no impact on P. aeruginosa adaptation. In contrast, CF-adapted S. aureus seemed to trigger the emergence of P. aeruginosa SCV, but the P. aeruginosa resistant strain was also able to produce SCV when grown alone in ASM. Therefore, both hypothesis were discarded which led to conclude that S. aureus seemed to have no role in P. aeruginosa pathogenicity in CF lungs. It was also concluded that the discrepancy of this study with literature may be explained by the different in vitro conditions used. The majority of the studies used standard laboratory medium that does not fully mimic the chemical and nutritional complex environment found in CF lungs. Moreover, this model used different inoculation timings of bacterial species to better mimic in vivo conditions, issue that was not took into account in the previous studies. 2-DE revealed some differences in protein expression by P. aeruginosa strains that could be involved in important biological processes, such as iron transport, carbon catabolism and lipids biosynthesis.