Date Published: June 4, 2019
Publisher: Public Library of Science
Author(s): Beata Maczynska, Anna Secewicz, Danuta Smutnicka, Patrycja Szymczyk, Ruth Dudek-Wicher, Adam Junka, Marzenna Bartoszewicz, Vernita D Gordon.
Biofilm-related infections of bones pose a significant therapeutic issue. In this article we present in vitro results of the efficacy of gentamicin released from a collagen sponge carrier against Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae biofilms preformed on hydroxyapatite surface. The results indicate that high local concentrations of gentamicin released from a sponge eradicate the biofilm formed not only by gentamicin-sensitive strains but, to some extent, also by those that display a resistance pattern in routine diagnostics. The data presented in this paper is of high clinical translational value and may find application in the treatment of bone infections.
It is believed that 60–80% of nosocomial infections are caused by biofilm pathogens. Therefore, detection and treatment of pathogenic biofilm is among the most significant healthcare issues . The extracellular matrix of biofilm contributes to its high tolerance to the host’s defense mechanisms, antibiotics and antiseptics [2,3]. Chronic infections of wounds and bones are also caused by biofilms [4–6]. The Gram-positive coccus, referred to as the Staphylococcus aureus, is considered the most ubiquitous etiological factor of such infections regardless its origin (nosocomial or community-acquired type). In turn, Gram-negative bacteria (Pseudomonas aeruginosa and Enterobacteriacae family members) occur more frequently in hospital-acquired infections [7–9]. Treatment of bone infections is a significant diagnostic and therapeutic problem. The specific anatomical structure of bone considerably limits the efficacy of antimicrobial measures and hinders the immune response [10–12]. Also, microbiological examination of bones is difficult due to problems with obtaining the appropriate diagnostic material. As regards treatment procedures, antibiotic therapy may raise some objections mostly because high doses of active agents (above the Minimum Inhibitory Concentration, MIC), required to be delivered to the infection site, may cause systemic toxicity . Implants saturated with gentamicin represent an important exception to the above rule. Numerous data indicate that the high concentration of gentamicin released locally from the implant does not contribute to the high systemic concentration of this antibiotic [12,14,15]. Gentamicin belongs to a class of antibiotics referred to as aminoglycosides, which are still commonly used to treat severe infections, especially in combination therapy. According to EARSS (European Antimicrobial Resistance Surveillance System) data from 2015 , resistance against aminoglycosides among Gram-negative rods P.aeruginosa and E.coli is still relatively low (30% and 11%, respectively). However, an upward trend is currently observed. Other Gram-negative rods, such as K.pneumoniae and Acinetobacter baumannii, display higher resistance frequencies (59% and 70%, respectively). Aminoglycosides have such functional advantages as rapid bactericidal effect [1-2h], post-antibiotic effect [PAE], inoculum-independent activity, synergy with beta-lactam and glycopeptide antibiotics as well as easy dosing (one dose/day) . On the other hand, there exist numerous microbial resistance patterns to aminoglycosides including enzymatic, receptor and transport mechanisms [17,18,19, 20]. Moreover, a systemic application of gentamicin in bone infection treatment is limited due to a low penetration ratio. Therefore, a number of approaches has been developed to increase this functional parameter. The most prominent of them include introduction of gentamicin with such natural carriers as albumins, collagens, chitosans, hyaluronic acids or with such synthetic carriers as polylactic acids, glycols, phosphates and hydroxyethylocellulose. All these approaches are designed to increase the antibiotic penetration through the biofilm and to allow a gradual release of the antimicrobial . Clinical data suggest that the following are the indications for the application of gentamicin sponge: osteomyelitis and other bone infections, prophylaxis during procedures at risk of infection (implantations, bone grafts, surgical procedures at infection sites), proctologic surgery and cardiac surgery including bridge infections . Purified I and III type collagen (from bovine tendons) is used in the gentamicin sponge [23, 24]. This natural polymer displays both low allergenicity and is biodegradable. Thus, as a carrier for gentamicin, a collagen sponge may be considered a fully biocompatible product. The biodegradation of the carrier eliminates the need of another surgery, accelerates wound healing and provides gradual and systematic gentamicin release [12,15]. It was previously demonstrated that gentamicin is released completely from the carrier during the first 60 min after implantation . The obtained concentrations exceeded the established MIC and reached the value of 1000mg/L. During the next 4–5 days after implantation, the antibiotic concentration was at the level of 300-400mg/L . However, in the serum, the measured gentamicin concentration was very low (below or equal to 2mg/L), which reduces the risk of systemic adverse effects, such as neuro- or nephrotoxicity) [12,15, 26]. Very high local concentration of the antibiotic suggests that also microorganisms of reduced sensitivity to gentamicin could be eradicated [18,19,20]. Therefore, the aim of this research was to evaluate the in vitro efficacy of high doses of gentamicin delivered locally via collagen sponge against bone pathogens.
Planktonic forms of the analyzed strains displayed diversified sensitivity to gentamicin. 85%, 71% and 41% of S.aureus, P.aeruginosa and K.pneumoniae strains, respectively, showed resistance to gentamicin using the standard E-test method. The results of microdilution method of antibiotic sensitivity estimation (also routinely used in microbiological diagnostics) were fully coherent with the E-test results in the case of S.aureus and P.aeruginosa but not for K. pneumoniae strains. In the case of this pathogen, 76% of the strains were considered resistant to gentamicin according to EUCAST guidelines. Next, the sensitivity of planktonic and biofilm forms (preformed on a polystyrene well of a 96-well plate) toward gentamicin was analyzed. When the S.aureus and P.aeruginosa strains were allowed to form biofilm, their tolerance to the antibiotic grew significantly in comparison to their planktonic counterparts (K-W test, p<0.05). In the case of Klebsiella pneumoniae, an analogical trend was visible, which was however statistically insignificant due to high standard deviations obtained [Fig 1]. Source: http://doi.org/10.1371/journal.pone.0217769