Identification and Morphological Characterization of Biofilms Formed by Strains Causing Infection in Orthopedic Implants

Abstract

Objectives: For a better understanding of the mechanisms involved in biofilm formation, we performed a broad identification and characterization of the strains affecting implants by evaluating the morphology of biofilms formed in vitro in correlation with tests of the strains' antibiotic susceptibility in planktonic form. The ability of the strains to form biofilms in vitro was evaluated by means of colony forming units counting, metabolic activity tests of biofilm cells, and scanning electron microscopy. Methods: A total of 140 strains were isolated from patients with orthopedic implant-related infections during the period of 2015 to 2018. The identification of the isolates was carried out through microbiological cultures and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility rates of the isolates were accessed according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). The ability of all isolates to form biofilms in vitro was evaluated by counting the colony forming units, by measuring the metabolic activity of biofilm cells, and by analyzing the morphology of the formed biofilms using scanning electron microscopy. Results: From all the isolates, 41.84% (62 strains) were Staphylococcus epidermidis and 15.60% (22 strains) were Staphylococcus aureus. A significant difference in the capacity of biofilm formation was observed among the isolates. When correlating the biofilm forming capacity of the isolates to their antibiotic susceptibility rates, we observed that not all strains that were classified as resistant were biofilm producers in vitro. In other words, bacteria that are not good biofilm formers can show increased tolerance to multiple antibiotic substances. Conclusion: From 2015 until 2018, Staphylococcus epidermidis was the strain that caused most of the orthopedic implant-related infections in our hospital. Not all strains causing infection in orthopedic implants are able to form biofilms under in vitro conditions. Differences were observed in the number of cells and morphology of the biofilms. In addition, antibiotic resistance is not directly related to the capacity of the strains to form biofilms in vitro. Further studies should consider the use of in vitro culture conditions that better reproduce the joint environment and the growth of biofilms in humans.

Keywords: antibiotic susceptibility; biofilm; implant-related infections.; in vitro conditions.

Figure 1

Clinical isolates obtained during the period of 2015 until 2018 from patients undergoing prosthetic joint infection (PJI) treatment.

Figure 2

Scanning electron microscopy of S. epidermidis biofilms isolated from patients undergoing PJI treatment. The images show massive (A,B), moderate (C,D), and slight (E,F) biofilm formation. Magnification for all images: 2000×. Specimens were analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany).

Figure 3

Scanning electron microscopy of S. aureus biofilms isolated from patients undergoing PJI treatment. The images show massive (A,B), moderate (C,D), and slight (E,F) biofilm formation. Magnification for all images: 2000×. Specimens were analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany).

Figure 4

Scanning electron microscopy of S. haemolyticus (A); S. oralis (B); P. avidum (C); S. lugdunensis (D); S. saprophyticus (E); B. casei (F); S. hominis (G); and S. capitis (H) biofilms isolated from patients undergoing PJI treatment. Magnification for all images: 2000×. Specimens were analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany).

Figure 5

Scanning electron microscopy of Corynebacterium glucuronolyticum (A); M. osloensis (B); MRSA (C); S. cohnii (D); S. simulans (E); and S. xylosus (F) biofilms isolated from patients undergoing PJI treatment. Magnification for all images: 2000×. Specimens were analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany).

Figure 6

Antibiotic susceptibility of S. epidermidis strains in relation to its biofilm forming capacity. The results of the susceptibility tests were classified as susceptible (green), intermediate (blue), and resistant (red) according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). Non-tested antibiotics are displayed in grey. The biofilm forming capacity was classified as no biofilm former (grey), low biofilm former (rose), and high biofilm former (dark red). The biofilm formation was classified according to the counting of colony forming units; measurement of metabolic activity; and morphology of the biofilms based on the scanning electron microscopy analysis.

Figure 7

Antibiotic susceptibility of S. aureus, S. capitis, and S. hominis strains in relation to their biofilm forming capacity. The results of the susceptibility tests were classified as susceptible (green), intermediate (blue), and resistant (red) according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). Non-tested antibiotics are displayed in grey. The biofilm forming capacity was classified as no biofilm former (grey), low biofilm former (rose), and high biofilm former (dark red). The biofilm formation was classified according to the counting of colony forming units; measurement of metabolic activity; and morphology of the biofilms based on the scanning electron microscopy analysis.

Figure 8

Antibiotic susceptibility of S. haemolyticus, S. warneri, M. luteus, S. oralis, P. avidum, S. lugdunensis, S. saprophyticus, B. fragilis, B. casei, C. glucuronolyticum, M. osloensis, MRSA, S. caprae, S. cohnii, S. pettenkoferi, S. simulans, and S. xylosus strains in relation to their biofilm forming capacity. The results of the susceptibility tests were classified as susceptible (green), intermediate (blue), and resistant (red) according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). Non-tested antibiotics are displayed in grey. The biofilm forming capacity was classified as no biofilm former (grey), low biofilm former (rose), and high biofilm former (dark red). The biofilm formation was classified according to the counting of colony forming units; measurement of metabolic activity; and morphology of the biofilms based on the scanning electron microscopy analysis.