Effect of Different Antibiotic Chemotherapies on Pseudomonas aeruginosa Infection In Vitro of Primary Human Corneal Fibroblast Cells

Abstract

Pseudomonas aeruginosa is a major cause of bacterial keratitis (BK) worldwide. Inappropriate or non-optimal antibiotic chemotherapy can lead to corneal perforation and rapid sight loss. In this study, we tested the hypothesis that P. aeruginosa strain PAO1 invades primary human corneal fibroblasts (hCFs) in vitro and persists intracellularly, despite chemotherapy with antibiotics used commonly to treat BK. In rank order, ciprofloxacin, levofloxacin and polymyxin B showed the highest activity against planktonic PAO1 growth (100% inhibitory concentration ≤10 μg/mL; 50% inhibitory concentration ≤1 μg/mL), followed by gentamicin and ofloxacin (100% inhibitory concentration ≤50 μg/mL; 50% inhibitory concentration ≤10 μg/mL). These bactericidal antibiotics (50–200 μg/mL concentrations) all killed PAO1 in the extracellular environment of infected hCF monolayers. By contrast, the bactericidal antibiotic cefuroxime and the bacteriostatic antibiotic chloramphenicol failed to sterilize both PAO1 broth cultures, even at a concentration of ≥200 μg/mL) and infected hCF monolayers. Statistically, all antibiotics were able to prevent LDH release from PAO1-infected hCF monolayers at both concentrations tested. Intracellular Pseudomonas were significantly reduced (>99%, P < 0.05) following treatment with ciprofloxacin, levofloxacin and ofloxacin, whereas gentamicin, polymyxin B and cefuroxime failed to clear intracellular bacteria over 24 h. Intracellular Pseudomonas infection was resistant to chloramphenicol, with hCF death observed by 9 h. Eventual growth of remaining intracellular Pseudomonas was observed in hCF after removal of all antibiotics, resulting in re-infection cycles and cell death by 48 h. All of the antibiotics reduced significantly (P < 0.05) IL-1β secretion by hCF infected with a Multiplicity Of Infection (MOI) = 1 of PAO1. With higher MOI, no pro-inflammatory effects were observed with antibiotic treatment, expect with polymyxin B and ofloxacin, which induced significant increased IL-1β secretion (P < 0.001). The findings from our study demonstrated that bactericidal and bacteriostatic antibiotics, routinely used to treat BK, failed to eradicate Pseudomonas infection of hCFs in vitro and that their bactericidal efficacies were influenced by the cellular location of the organism.

Differential adaptability between reference strains and clinical isolates of Pseudomonas aeruginosa into the lung epithelium intracellular lifestyle

“…Furthermore, the CFTR protein has been postulated to be important for P. aeruginosa internalization inside lung epithelial cells [ 25 ], which supports the idea that the clinical CF isolates PAET1, PAET2, and PAET4 may have undergone directed adaptation to allow them to colonize CFBE41o- cells, as we have detected in our work. Inside the cell, P. aeruginosa can persist and replicate intracellularly, as detected in our study as well as in other cellular models [ 7 , 17 , 25 ]. Bacterial persistence within host cells can constitute a latent reservoir that is helped by the poor intracellular penetration of some antibiotics and can lead to the selection of antibacterial resistant mutants and contribute to disease harshness and chronicity [ 7 , 21 ].…”

“…However, the invasion rates and the levels of intracellular persistence and replication were found to be cell- and strain-dependent. The invasion capacity of the P. aeruginosa strains within host cells in vitro notwithstanding the P. aeruginosa phenotype have been broadly observed [ 7 , 17 , 21 , 23 ]. It should be noted that there is heterogeneous genotypic clonality among P. aeruginosa infections as well as different protein expression profiles (including important virulence factors) that depend on the P. aeruginosa background [ 4 , 24 ].…”

“…Infection with the different P. aeruginosa strains was performed at a multiplicity of infection (MOI) of 100 in antibiotic-free DMEM for 3 h. To determine bacterial adhesion (adhesion and invasion), cell monolayers were gently washed three times with warm 1x phosphate-buffered saline (PBS; pH 7.4) and lysed with 200 µl of PBS containing saponin (0.1% w/v; saponin buffer) for 15 min. To quantify the intracellular invasion of the different P. aeruginosa strains, we used the gentamicin survival assay as previously described [ 17 ]. Briefly, after 3 h of infection, monolayers were washed with warm 1X PBS and then incubated with DMEM containing gentamicin 200 µg/mL (gentamicin solution) for 90 min to kill extracellular P. aeruginosa .…”

“…P. aeruginosa phenotype have been broadly observed [7,17,21,23]. It should be noted that there is heterogeneous genotypic clonality among P. aeruginosa infections as well as different protein expression profiles (including important virulence factors) that depend on the P. aeruginosa background [4,24].…”

Management of Psuedomonas Keratitis: a review article

“…It was also found that bactericidal and bacteriostatic antibiotics used in bacterial keratitis were unable to eradicate Pseudomonas infection of human fibroblasts culture in vitro. Their effectiveness depends on the cellular location of Pseudomonas [36].…”