Bacterial infection of wounds is a major risk for patients undergoing skin grafts following severe burn injuries. Drs Monica Iliescu Nelea (left) & Michel Alain Danino, of the Plastic and Reconstructive Surgery Department at the University of Montreal Hospital Center (CHUM), Montreal, Canada are part of a group of researchers working on furthering medical understanding of this phenomenon.
A biofilm is a layer of bacterial microorganisms that have aggregated to form a colony. Biofilms can develop on many surfaces, including wound dressings. This can be particularly critical for patients undergoing skin grafting for burn wounds since they often suffer from a weak or compromised immune system.
The areas where healthy skin is taken from, known as donor sites, are particularly vulnerable to biofilm formation, the presence of which strongly compromises normal wound healing. This in turn can become a source of pain for the patient and ultimately affects scar aesthetics.
The SEM: a state-of-the-art tool for biomaterial surface characterization
Using the Thermo Scientific™ (former FEI) Quanta™ 450 FEG Environmental Scanning Electron Microscope (ESEM™), researchers compared the efficacy of two types of wound dressings used in preventing the formation of bacterial biofilm on burn patient skin graft donor sites. One dressing contained bismuth tribromophenate at a concentration of 3% which confers it bacteriostatic (antibacterial) properties (Xeroform™). The other was an absorptive alginate calcium sodium dressing (Kaltostat™).
Dressing samples derived directly from actual burn patient donor sites were analyzed for bacterial biofilm density and then compared. The samples were prepared for analysis under the scanning electron microscope (SEM) using an original method developed by the research team that aims to maintain the integrity of the biofilm microstructure. To this day, SEM imaging has rarely been employed for dressing analysis and this is the first time that it has been used for in-situ biofilm visualization for this particular application.
In the SEM images obtained of the wound dressing samples, the research team assigned a score based on the predominance of bacteria and the presence of bacterial biofilm.
Above. Colorized SEM images of the Kaltostat™ wound dressing at different magnifications. Colorization was performed with TopoMaps software based on Mountains®. Bacterial biofilms are made visible by colorization in light brown.
Different types of bacteria were easily identifiable in the SEM images, both in isolated forms and in clusters within biofilm. For example, it was possible to observe cocci-type bacteria establishing biofilm. Bacilli-type biofilm and streptococci were also observed. All these types of bacteria were detected for both dressings, the main difference being that they were highly visible in the Kaltostat™ dressing and were less notable in both quantity and frequency in the Xeroform™ dressing.
The images above, colorized using Mountains®- based TopoMaps software1, attest to the presence and extent of bacterial biofilm in a lower magnification view (A) where bacterial biofilm is colored in brown and wound dressing fibers in blue.
Furthermore, a higher magnification view image (B) shows bacterial clusters (colored in brown) as well as the extracellular polysaccharide network (EPS matrix, colored in green) which partially surrounds the bacteria, forming the biofilm. “Colorizing the images helped us to better highlight the presence of bacterial biofilms on SEM images at low magnification” stated Dr Iliescu, “It also helped to enhance the presence of bacterial aggregates (clusters) and the formation of the EPS matrix on SEM images at high magnification.”
Further possibilities in biomedical research
This study allowed the team to show that the scanning electron microscope is undoubtedly an immensely valuable tool for the analysis of wound dressing samples for which it had not been previously used.
This imaging technique permits researchers to visualize and characterize bacterial biofilms, provided that the preparation technique is appropriate and that results are correctly analyzed.
The team was able to observe that while donor sites covered with the commonly used wound dressing Kaltostat™ often became breeding grounds for microorganisms, the dressing with bacteriostatic properties (Xeroform™) hindered bacterial proliferation and accelerated wound healing. In other words, bacterial biofilm formation was more pronounced in the wound dressing without bacteriostatic properties.
▶ In-situ characterization of the bacterial biofilm associated with Xeroform™ and Kaltostat™ dressings and evaluation of their effectiveness on thin skin engraftment donor sites in burn patients. Monica Iliescu Nelea, Laurence Paek, Lan Dao, Nathalie Rouchet, Johnny I. Efanov, Coeugniet Édouard, Michel A. Danino. Burns, Volume 45, Issue 5, August 2019, Pages 11221130. doi.org/10.1016/j.burns.2019.02.024
Colorize your SEM images in just a few clicks
Quick & easy SEM image colorization explained in this video:
Instruments & software used
Thermo Scientific™ (former FEI) Quanta™ 450 FEG Environmental Scanning Electron Microscope (ESEM™) + TopoMaps software
Maambukizo ya bakteria ya majeraha ni hatari kubwa kwa wagonjwa wanaopatiwa upasuaji wa ngozi baada ya majeraha makubwa ya kuchomwa moto. Drs Monica Iliescu-Nelea na Michel Alain Danino, wa Idara ya Upasuaji wa Plastic na Reconstructive katika Chuo Kikuu cha Montreal Hospital Center (CHUM), Montreal, Canada, ni sehemu ya kikundi cha watafiti wanaofanya kazi ya kuendeleza uelewa wa matibabu wa jambo hili. Biofilm ni safu ya viumbe wadogo wa bakteria ambao wameungana ili kuunda koloni. Filamu za kibiolojia zaweza kuenea kwenye nyuso nyingi, kutia ndani vifuniko vya majeraha. Hii inaweza kuwa muhimu hasa kwa wagonjwa wanaopitia upasuaji wa ngozi kwa majeraha ya kuchoma kwani mara nyingi wanateseka kutokana na mfumo dhaifu wa kinga au ulioharibiwa. Sehemu ambazo ngozi ya afya inachukuliwa kutoka, inayojulikana kama maeneo ya wafadhili, ni hatari sana kwa malezi ya biofilm, uwepo wake unahatarisha sana uponyaji wa kawaida wa jeraha. Hilo laweza kuwa chanzo cha maumivu kwa mgonjwa na hatimaye kuathiri urembo wa kidonda. Kwa kutumia Thermo Scientific (FEI) Quanta 450 FEG (Environmental Scanning Electron Microscope) watafiti walilinganisha ufanisi wa aina mbili za bandages za jeraha zinazotumiwa kuzuia kuundwa kwa biofilm ya bakteria kwenye maeneo ya wafadhili wa ngozi ya mgonjwa wa kuchoma. Kwa mfano, dawa ya kuondoa ugonjwa wa kisukari ya Bismuth-tribromophenate (BTP) ina uwezo wa kuondoa bakteria na kuondoa bakteria. Mto huo ulikuwa na dawa ya kuingiza alginate ya kalsiamu na sodiamu (KaltostatTM). Sampuli za kuvalia zilizopatikana moja kwa moja kutoka kwa maeneo halisi ya wafadhili wa wagonjwa wa kuchoma zilichambuliwa kwa wiani wa biofilm ya bakteria na kisha kulinganishwa. Sampuli hizo zilitayarishwa kwa ajili ya uchambuzi chini ya darubini ya elektroniki ya kuchunguza (SEM) kwa kutumia mbinu ya awali iliyoundwa na timu ya utafiti ambayo inakusudia kudumisha uadilifu wa biofilm microstructure. Kwa sasa, SEM imewekwa katika matumizi ya uchunguzi wa biofilm, na kwa mara ya kwanza inatumika kwa ajili ya uchunguzi wa biofilm. Katika picha za SEM zilizopatikana kutoka kwa sampuli za kuvalia jeraha, timu ya utafiti ilipewa alama kulingana na utajiri wa bakteria na uwepo wa biofilm ya bakteria. Juu ya hapo. Picha za rangi za SEM za Kaltostat TM za kufunika jeraha kwa kuongeza tofauti. Rangi ilifanywa na programu ya TopoMaps kulingana na Milima®. Biofilms za bakteria huonekana kwa rangi ya kahawia nyepesi. Aina tofauti za bakteria zilitambuliwa kwa urahisi katika picha za SEM, kwa namna za pekee na kwa makundi ndani ya biofilm. Kwa mfano, iliwezekana kuchunguza bakteria za aina ya cocci zikianzisha biofilm. Biofilm ya aina ya bacilli na streptococci pia zilionekana. Aina zote za bakteria ziligunduliwa kwa dressings zote mbili, tofauti kuu ni kwamba zilionekana sana katika dressing ya Kaltostat na zilikuwa chini ya kuonekana katika wingi na mzunguko katika dressing ya Xeroform. Picha zilizo juu, zilizochorwa kwa kutumia programu ya TopoMaps ya Milima, zinathibitisha uwepo na kiwango cha biofilm ya bakteria katika mtazamo wa upanuzi wa chini (A) ambapo biofilm ya bakteria ni rangi ya kahawia na nyuzi za kuvalia jeraha kwa bluu. Picha ya juu ya upanuzi (B) inaonyesha makundi ya bakteria (rangi ya kahawia) na pia mtandao wa polysaccharide wa nje ya seli (matrix ya EPS, rangi ya kijani) ambayo inazunguka bakteria kwa sehemu, kuunda biofilm. "Kutumia rangi ya picha ilituwezesha kuonyesha vizuri uwepo wa biofilms ya bakteria kwenye picha za SEM kwa kuongeza kidogo, ""alisema Dk Iliescu, ""Ilikusaidia pia kuongeza uwepo wa vifurushi vya bakteria na kuundwa kwa matrix ya EPS kwenye picha za SEM kwa kuongeza kubwa." Mbinu hii ya picha inaruhusu watafiti kuona na kuelezea biofilms za bakteria, ikiwa mbinu ya maandalizi ni sahihi na matokeo yanachambuliwa kwa usahihi. Wakati maeneo ya wafadhili yaliyofunikwa na bandage ya jeraha ya kawaida ya Kaltostat mara nyingi huwa mahali pa kuzaliana kwa viumbe wadogo, bandage yenye mali ya bakteria (Xeroform) ilizuia kuenea kwa bakteria na kuharakisha uponyaji wa jeraha. Kwa maneno mengine, uundaji wa biofilm ya bakteria ulikuwa wazi zaidi katika kufunika jeraha bila mali ya bacteriostatic. In-situ characterization ya biofilm bakteria kuhusishwa na XeroformTM na KaltostatTM bandages na tathmini ya ufanisi wao juu ya nyembamba ngozi engraftment wafadhili maeneo katika wagonjwa kuchoma. Monica Iliescu Nelea, Laurence Paek, Lan Dao, Nathalie Rouchet, Johnny I. Efanov, Coeugniet Édouard, Michel A. Danino. "Kutoka kwa ""SEM"" (SEM) - ""SEM"" (SEM) - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""SEM"" - ""S"" - ""S"" - ""S"" - ""S"" - ""S"" - ""S""