Drug Synergistic Effects of Coriandrum sativum, Piperacillin, and Tazobactam Against Biofilms of Thioredoxin-A Protein Mutant Strains of Acinetobacter baumannii

Introduction:: Acinetobacter baumannii (AB), a Gram-negative opportunistic pathogen is recognized by The Centers for Disease Control and Prevention (CDC) and The World Health Organization (WHO) as one of the most notorious microorganisms to treat globally, especially within healthcare settings. Respiratory, urinary, and wound infections are commonly reported with AB. Immunocompromised individuals and military soldiers are most prone to such infections. The notorious nature of the microbe can be attributed largely to its ability to act as a Multi-Drug Resistant (MDR) bacterium. In a 2010 study, greater than 50% of AB clinical isolates were classified as resistant to greater than three classes of antibiotics. AB’s nosocomial infections lead to increased hospitalization costs and are associated with mortality rates greater than 50%. The ability of AB to resist antibiotics is a result of intertwined complex mechanisms that rely on expression of virulence genes, secretion of certain proteins, utilization of efflux pumps, biofilm formation as well as survival in harsh environmental conditions. Of particular interest to us is the role of the Thioredoxin-A (Trx-A) system in controlling biofilm formation and MDR. Trx-A protects cells against reactive oxygen species and controls the structure and activity of proteins that contain regulatory cysteines and thiol-dependent proteins through manipulations to cell surface hydrophobicity (CSH). As such, we hypothesize that by targeting the hydrophobic domains of AB strains, effective antibiotics that are capable of eradicating AB biofilms and controlling AB infections can be identified.

Materials and Methods:: Four AB strains that include two wild types (WT) and two mutants with Trx-A genes deleted were investigated (5075WT, 5075DTrxA, Ci790WT, and Ci79DTrxA). The strains were cultured at 37 ^oC and 150 rpm in Luria Bertani (LB) broth until late exponential phase of growth. The cells were treated in a range of concentrations with Tazobactam (TAZ), b-lactamase inhibitor, Piperacillin (PIP), a b-lactam, and C. sativum (CS)(an herbal plant), individually or synergistically. For all strains, the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) were determined. Contact angle measurements of wettability and electrophoretic mobilities measurements as a function of ionic strength and antibiotic exposure were carried out in a goniometer and zeta sizer according to our published protocols¹. Soft-particle analysis of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was used to predict the likelihood of AB to form biofilms on a variety of hospital materials. All measurements were done in triplicates. One-Way ANOVA was used to compare results.


Results, Conclusions, and Discussions:: Results and Discussion: Prior to AB’s exposure to antibiotics, all AB strains were hydrophilic. Upon exposure to TAZ to TAZ-CS at MIC concentrations (32 mg/mL each), the WT strains remained hydrophilic while the mutant DTrx-A strains became hydrophobic. Our checkerboard assay results indicated that the use of PIP alone or in combination with TAZ for up to a concentration of 200 mg/mL was not effective against any of the AB strains. In comparison, the use of TAZ alone suppressed the biofilm formation of all AB strains at 200 mg/mL and the Ci79 WT and DTrxA strains at 32 mg/mL. With the above in mind, we have decided to further explore the efficacy of TAZ or TAZ-CS at 32 mg/ml each against AB biofilms in a time kill assay in which the antibiotic is refreshed every 12 hours (Figure 1). Our findings indicated that exposing the Trx A mutant strains to either antibiotic over a period of 30 hours resulted in over a 3-log reduction in growth (Figures 1 B and 1D); signifying that these strains are susceptible. In comparison, the WT strains did not show a 3-log reduction in growth for any treatment or for any time point (Figures 1 A and 1C). The susceptibility of the mutant strains to TAZ and TAZ-CS can be attributed to the lack of Trx A in the mutants. When results were modeled using SOFT-DLVO, our results indicated that the untreated AB strains have attractive energies to Teflon, Polyethylene, and Polyvinylchloride; all common hospital materials.





Conclusions: The strains investigated varied in their properties with DTrxA strains being more hydrophobic than the WT strains; confirming the importance of Trx-A system in controlling cellular properties and biofilm formation. Our results indicated that the use of 32 μg/mL of TAZ and 32 μg/mL of TAZ-CS were highly effective towards the mutant strains compared to the WT strains. Overall, our results suggest that TAZ and TAZ-CS should be explored as novel treatment options for AB infections.


Acknowledgements (Optional): : The AB strains were supplied through collaboration with Dr. Jieh-Juen Yu at The University of Texas at San Antonio.


References (Optional): : 1) A.O. Eskhan and N.I. Abu-Lail, Force-averaging DLVO model predictions of the adhesion strengths quantified for pathogenic Listeria monocytogenes EGDe grown under variable pH stresses, Langmuir, 2020, 36, 30, 8947–8964.