Prevention and Killing Efficacy of Carbapenem Resistant Enterobacteriaceae (CRE) and Vancomycin Resistant Enterococci (VRE) Biofilms by Antibiotic-Loaded Calcium Sulfate Beads.

Paul Stoodley,Nan Jiang,Craig P Delury,Sean S Aiken,Devendra H Dusane,Phillip A Laycock,Casey W Peters,Jacob Brooks

doi:10.3390/ma13153258

Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE) have emerged as multidrug-resistant (MDR) pathogens associated with periprosthetic joint infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ALCSB) in inhibiting bacterial growth, encouraging biofilm formation and killing preformed biofilms of CRE and VRE. Three strains of Klebsiella pneumoniae (KP) and a strain of Enterococcus faecalis (EF) were used. ALCSB of 4.8-mm diameter were loaded with vancomycin (V) and gentamicin (G), V and rifampicin (R), V and tobramycin (T) or R and meropenem (M), and placed onto tryptic soy agar (TSA), spread with one of the test strains and incubated for 24 h at 37 °C. Beads were transferred daily onto fresh TSA spread plates and the zone of inhibition (ZOI) was recorded until no inhibition was observed. ALCSB containing R + M or R + V produced the most extensive ZOI up to 5 weeks. Biofilm prevention efficacy was investigated by challenging ALCSB daily with 5 × 105 CFU/mL bacterial cells and analyzing for biofilm formation at challenges 1, 2 and 3. In the biofilm killing experiments, ALCSB were added to pre-grown 3-day biofilms of KP and EF strains, which were then analyzed at days 1 and 3 post-exposure. The CFU counts and confocal images of the attached cells showed that ALCSB treatment reduced colonization and biofilm formation significantly (5–7 logs) with combinations of R + M or R + V, compared to unloaded beads. This study provides evidence that the local release of antibiotics from ALCSB may be useful in treating the biofilms of multidrug-resistant strains of CRE and VRE.

Highlights

In recent years, multidrug-resistant organisms, especially vancomycin-resistant enterococcus (VRE) and carbapenem-resistant enterobacteriaceae (CRE) strains have become more prevalentMaterials 2020, 13, 3258; doi:10.3390/ma13153258 www.mdpi.com/journal/materialsMaterials 2020, 13, 3258 worldwide, and represent a serious public health threat since they are becoming increasingly resistant to current treatment modalities [1]
Biofilms are microbial populations that adhere to surfaces forming a community inherently highly tolerant to antibiotics compared to their planktonic counterparts, even if isolates are shown to be sensitive by routine clinical microbiological methods, such as the minimum inhibitory concentration (MIC) assay [11]
There is little information concerning whether carbapenem-resistant Klebsiella pneumoniae and vancomycin-resistant E. faecalis strains form biofilms, or how they might be killed by antibiotic-loaded calcium sulfate beads (ALCSB)

Summary

Introduction

Multidrug-resistant organisms, especially vancomycin-resistant enterococcus (VRE) and carbapenem-resistant enterobacteriaceae (CRE) strains have become more prevalentMaterials 2020, 13, 3258; doi:10.3390/ma13153258 www.mdpi.com/journal/materialsMaterials 2020, 13, 3258 worldwide, and represent a serious public health threat since they are becoming increasingly resistant to current treatment modalities [1]. Whilst Staphylococci are the most common PJI pathogens, VRE and carbapenem-resistant Klebsiella pneumoniae strains (a CRE strain) have been reported, and are associated with poor outcomes [9,10]. Treatment strategies for PJI in total hip and total knee arthroplasty patients include the use of bone cement [14,15], and absorbable calcium sulfate void fillers loaded with antibiotics [16,17], to increase the local concentration and exposure period at the surgical site, beyond those obtainable with systemic administration [18]. In vitro studies show how antibiotic-loaded calcium sulfate beads (ALCSB) can significantly reduce or even eradicate bacterial biofilms of various Gram-positive and -negative PJI pathogens [11,19,20]. The goals of the present study were as follows: (i) to assess biofilm formation in three CRE strains (including the metallo-beta-lactamase New Delhi strain) and one VRE strain, and (ii) to quantify the long-term bioactivities of various combinations of antibiotics released from ALCSBs, in order to determine the ability to prevent biofilm formation or kill pre-established biofilms of these strains in vitro

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