Inhibitory activity of Chlorogenic Acid and Coffee Silverskin extracts against Klebsiella pneumoniae carbapenemases.

Coffee processing produces numerous waste by-products (around two billion tonnes), including silverskin (SS). A possible reuse of these by-products is desirable, as they are not only an environmental but also an economic burden. Based on the observation that chlorogenic acid has anti- Extended-Spectrum-β-Lactamase activity [1] and on the presence of this compound in SS [2], the aim of this work was to evaluate the inhibitory activity of chlorogenic acid and SS extracts against Klebsiella pneumoniae type-two carbapenemase (KPC2). Using four extraction solvents, H2O, MeOH, MeOH:H2O (50:50) and EtOH:H2O (70:30), four SS extracts were obtained. The inhibition activity of the carbapenemase-dependent resistance mechanism was measured against Klebsiella pneumoniae clinical strains KP-187 (KPC2-positive), KP-5 (KPC2-positive) and KP-ATCC13823 (carbapenem-susceptible control strain). Escherichia coli ATCC25922 was used as tester strain. In all experiments, imipenem was the indicator carbapenem antibiotic. To detect anti-carbapenemase activity, an adaptation of the Carbapenem Inactivation Method (CIM) [3] was developed and referred to as reverse Carbapenem Inactivation Method (rCIM). The protocol requires that produced carbapenemases are released from the cells after lysis, which was achieved by suspending 10 μl of bacterial culture in 200 μl of a Lysis Buffer. A disc preloaded with imipenem with or without putative inhibitors (i.e. chlorogenic acid or SS extracts) was added to the cell lysate. The highest concentrations of chlorogenic acid and extracts were 25 μg/ml and 500 μg/ml, respectively. After incubation at 37°C for 20 minutes, the disc was placed on an agar plate previously seeded with a 3 McFarland units suspension of the carbapenem-susceptible E. coli tester strain. Incubation at 37°C followed and results were recorded at two endpoints: 3 hours and 24 hours. The presence of a halo around the disc was an indication that the carbapenemase was not present or inhibited. To exclude that lack of carbapenemase activity could depend on inefficient bacterial cell lysis, experiments were carried out using a modified Lysis Buffer, wherein the concentration of the components with lytic activity was doubled. The SS extracts and chlorogenic acid showed no carbapenemase inhibitory activity irrespective to the Lysis conditions applied. Contrary to the observation against extendedspectrum beta lactamases (ESBL), pure chlorogenic acid did not show activity against KPC2 under the experimental conditions used. Also SS extracts did not inhibit the enzyme.