MICROSCOPY RESEARCH AND TECHNIQUE 77:305–312 (2014)
Endotracheal Tube Biofilm and Ventilator-Associated Pneumonia With Mechanical Ventilation PAULA REGINA DE SOUZA,1 DENISE DE ANDRADE,2 DANIELLE BEZERRA CABRAL,2 AND EVANDRO WATANABE3* 1
Department of Nursing, University of Brasılia, Brasılia, Distrito Federal, Brazil Department of General and Specialized Nursing, University of S~ ao Paulo at Ribeir~ ao Preto College of Nursing, Ribeir~ ao Preto, S~ ao Paulo, Brazil 3 Department of Restorative Dentistry, University of S~ ao Paulo, School of Dentistry of Riber~ ao Preto, Ribeir~ ao Preto, S~ ao Paulo, Brazil 2
KEY WORDS
biofilms; pneumonia; ventilator-associated; intubation; intratracheal; crossinfection
ABSTRACT Objective: To analyze biofilm on internal and external surfaces of endotracheal tubes after their use in critical care patients, and to produce evidence of association between use of the tube, presence of biofilm, and the occurrence of pneumonia. Methods: This was a clinical study performed at the Intensive Care Unit of an emergency hospital in the interior of S~ ao Paulo state, Brazil. Data collection involved 30 endotracheal tubes used on adult patients for a period of 48 h of mechanical ventilation for scanning electron microscopy. Results: Analysis of the biofilm on the 30 tubes by scanning electron microscopy showed various abiotic and biotic structures, predominately on the internal surface, such as: fibrin network, erythrocytes, leukocytes, cocci, bacilli, and molds, among others. The intubation period of the endotracheal tube for 8 days represented one of the risk factors for ventilator-associated pneumonia (RR 7.41, P < 0.001). Conclusions: The presence of the endotracheal tube permits microbial colonization, overall contributing to the development of biofilm and the occurrence of pneumonia. Microsc. Res. Tech. 77:305–312, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Ventilator-associated pneumonia (VAP) is the most frequent hospital infection in intensive care units (ICUs), resulting in prolonged length of stay, higher rates of morbimortality, and a significant increase of costs (Bekaert et al., 2011; Melsen et al., 2011; Nair and Niederman, 2013). Patients who undergo mechanical ventilation have a risk of 10–65% of developing pneumonia, and 24–76% risk of mortality, depending on the research method and sample as well as the therapy adopted (Augustyn, 2007; Barbier et al., 2013). The diagnosis of pneumonia should be confirmed starting at 48 h of mechanical ventilation (Masterton et al., 2007; Mietto et al., 2013; Tablan et al., 2004). The presence of the endotracheal tube is an important causal factor in the genesis of VAP, because it modifies the anatomy of the larynx, causing damages to the mucociliary system, and acting as an entry point for microorganisms to the lower airways, through microbial adherence and multiplication on the tracheal surface (biofilm) (Pinciroli et al., 2013). Biofilm is considered the new paradigm of infection, especially associated on implants of prostheses, tubes, or catheters, for example, the endotracheal tube. The presence of the endotracheal tube undoubtedly favors microbial colonization, contributing overall to the development of biofilm, which may explain the occurrence of pneumonia (Hall-Stoodley et al., 2004; Kubota et al., 2008; Schaudinn et al., 2007). Biofilm is a microbial complex, embedded in a matrix of extracellular polymeric substances (EPS), designed on C V
2014 WILEY PERIODICALS, INC.
various surface types, whether abiotic such as rocks, metals and polymers, or biotic such as vegetable and animal tissues. Further, the formation of microenvironments on the biofilm offers protection to this microbial community against biological, physical, and chemical agents from the environment (Costerton et al., 1995; Maddula et al., 2006; Santos et al., 2011). In general, studies support the theory that the formation of biofilm can be an important mechanism of tube obstruction, in addition to representing a potential source of lung infection. Added to this, studies suggest that there is a relation between the use time of the tube and production of respiratory secretions, primarily in the subglottic region (Bassetti et al., 2012; Ramirez et al., 2007; Smulders et al., 2002). Studies have suggested using scanning electron microscopy (SEM) to analyze the biofilm, whose image provides unique advantages such as high resolution and large depth of field, which allows a detailed description of the topography of biofilms and their *Correspondence to: Evandro Watanabe, Av. do Cafe, s/no, Monte Alegre, Ribeir~ ao Preto, S~ ao Paulo, Brazil. E-mail: [email protected] Received 10 September 2013; accepted in revised form 24 January 2014 REVIEW EDITOR: Prof. George Perry Part of doctoral thesis, titled “Microbiological molecular-genetic analysis of the orotracheal biota of critical care patients: supports in the prevention of ventilator-associated pneumonia.” Contract grant sponsor: Brazilian funding agencies FAPESP and CAPES (Ph.D. fellowship). DOI 10.1002/jemt.22344 Published online 11 February 2014 in Wiley Online Library (wileyonlinelibrary. com).
306
P.R. DE SOUZA ET AL.
Fig. 1. SEM-generated photomicrographs of the internal (left side) and external (right side) surfaces of the sterilized endotracheal tube without the presence of biofilm.
TABLE 1. Morphological characteristics of biofilm on the 30 endotracheal tubes (internal and external surfaces) used on patients submitted to mechanical ventilation Internal surface Characteristics EPS matrix Compacted Fragmented Honeycomb Erythrocytes Fibrin network Leukocytes Cocci Microbial interaction Mold Bacilli
Yes No Yes No Yes No Yes No Yes No Yes No Yes No
External surface
n
%
n
%
22 4 4 3 7 5 25 5 25 1 29 1 29 1 2 0 30
73.3 13.3 13.3 10.0 90.0 16.7 83.3 16.7 83.3 3.3 96.7 3.3 96.7 3.3 96.7 0 100
12 17 1 2 28 1 29 2 28 0 30 0 30 0 30 1 29
40.0 56.7 3.3 6.7 93.3 3.3 96.7 6.7 93.3 0 100 0 100 0 100 3.3 96.7
n 5 number of samples; EPS 5 extracellular polymeric substances.
structure (Baldasso et al., 2012; Inglis, 1989, 1995; Schaudinn et al., 2007; Sottile et al., 1986). The aim of this study was to analyze the biofilm on internal and external surfaces of endotracheal tubes after their use on critical care patients, and to produce evidence of the association between use of the tube, presence of biofilm, and the occurrence of pneumonia. MATERIALS AND METHODS This study had a clinical focus, and was performed at the adult ICU of a public teaching hospital in the interior of the state of S~ ao Paulo, Brazil. The research was approved by the Research Ethics Committee (protocol 987/2008) of the studied hospital, and involved 30 study subjects of both sexes, aged between 18 and 60 years, who were critical care patients with endotracheal tubes who underwent mechanical ventilation for a period of 48 h of tracheal
intubation. Patients who developed pneumonia within 48 h of admission to the ICU as well as those with accidental extubation and intubation at another hospital were excluded from this study. Following extubation by physician recommendation, the endotracheal tubes were placed in sterilized, separate plastic bags, in a class II biological safety cabinet, for preparation of the samples and later analysis by SEM (Silveira, 2007). Sample preparation consisted of the process of dehydration with ethyl alcohol in different concentrations of 30, 50, 70, 90, and 95%, sequentially, for a period of 10 min, and 100%, three times, for 20 min. After dehydration, proceeded drying with CO2 (BalTec CPD 030, Critical Point Dryer—Furstentem—Liechtenstein), mounting on metal supports with a conductive adhesive (Colloidal Graphite Isopropanol Base), and vacuum metallization in gold for analysis of the surfaces of the endotracheal tubes by SEM. In this research, 30 fragments of endotracheal tubes (internal and external surfaces) used on critical care patients were evaluated, totaling 112 SEM-generated photographic records. In this research, biofilms were analyzed by scanning electron microscope (SEM) and a sterilized endotracheal tube was used as negative control. Moreover, all molecules and microorganisms were morphologically identified and EPS matrix structures were observed and classified as described by Donlan and Costerton (2002) and Schaudinn et al. (2007). The physician diagnosed cases of VAP based on clinical and radiological examination in accordance with CDC criteria (Tablan et al., 2004). Analysis of the association between potential predictors (endotracheal tube intubation period and outcome (ventilator-associated pneumonia) and agreement between the different measurements was performed through bivariate contingency tables. RESULTS AND DISCUSSION To evaluate the presence of biofilm on the samples, an unused, sterilized endotracheal tube was used as a negative control, which presented irregularities on the Microscopy Research and Technique
ENDOTRACHEAL TUBE BIOFILM AND VENTILATOR-ASSOCIATED PNEUMONIA
307
Fig. 2. SEM-generated photomicrographs of the endotracheal tubes with biofilm, compact EPS matrix (up side) and fragmented EPS matrix (down side).
internal and external surfaces, characteristics of the material that can favor the development of biofilm, as observed in Figure 1. All the samples presented biofilm, and the morphological characteristics are shown on Table 1. According to Table 1, the presence of the compacted and fragmented EPS matrix is more evident on the internal and external surfaces of the endotracheal tubes, respectively, as the internal surfaces of the tubes show less mechanical friction than the external, in direct contact with the trachea of the patient (Fig. 2). It was observed that the biofilm on the distal surfaces (internal and external) of the endotracheal tubes had diversified structures, not only in terms of morphological characteristics of the microbiota but also in the composition of elements such as fibrin, erythrocytes, and leukocytes, as shown in Table 1. In other words, it should also be considered that there is a direct relationship between use time of devices and the formation of biofilm, and that the use time represents an important factor in the occurrence of infection. It is important to note that the presence of biofilm on the endotracheal tubes of critical care patients, independent of the occurrence or not of VAP, reinforces the data from the literature that microorganisms Microscopy Research and Technique
adhere to the biomaterials, forming a microbial community on these surfaces (Berra et al., 2008; Chambless et al., 2006; Costerton et al., 1995; Hall-Stoodley et al., 2004). The EPS produced by microorganisms are constituted of different substances, intermediate microbial adherence to the surface of the material, and constitute the biofilm matrix. This matrix corresponds to approximately 85% of the volume of the biofilm (Donlan and Costerton, 2002). Furthermore, a structure resembling a “honeycomb,” documented in Figure 3 and described by Schaudinn et al. (2007), was clear on the external and internal surfaces of three endotracheal tubes. The honeycomb structure was observed in biofilm produced by Staphylococcus epidermidis and Pseudomonas spp., showing a tridimensional aspect with six elastic, symmetrical axes that confer flexibility and stability, permitting deformity to occur in response to stress, and subsequent return to the original shape. This characteristic endows the biofilm with greater resistance to adversity from the environment as well as antimicrobial action, considered an important virulence factor. It also favors multidirectional flow throughout the extension of the biofilm, allowing for less energy expense, and greater area of nutrient absorption (Schaudinn et al., 2007).
308
P.R. DE SOUZA ET AL.
Fig. 3. SEM-generated photomicrographs of endotracheal tubes with biofilm in the shape of a “honeycomb.”
Considering that the biofilm is constituted of approximately 90–95% water, during drying (critical point), a preparation stage of the sample for SEM, a substantial proportion of the volume of this biofilm is eliminated. This dehydration provokes the destruction of the structural architecture of the biofilm. Thus, the SEM tends to emphasize the microorganisms, sometimes observed in openings or fissures in the biofilm (Inglis, 1995; Schaudinn et al., 2007; Sottile et al., 1986). Thus, a concerning result was the proven presence of filamentous fungi and yeast (26.6%), bacilli (13.3%), cocci (6.6%), and microbial interactions (6.6%) on the endotracheal tubes (Table 1, Figs. 4–7). The bacteria commonly isolated from endotracheal tube biofilms include: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus intermedius, Streptococcus viridans, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Added to these are the following species of coagulasenegative Staphylococcus: Staphylococcus capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus auricularis, and Staphylococcus carnosus (Barbier et al., 2013; Feldman, 1999; Kojic and Darouiche, 2004; Otto, 2008). The microbial interactions that permit the coaggregation of different species, or even microbial
genera in a biofilm, generally involve the participation of adhesins (adherence molecules present in fimbriae or dispersed along the cell surface), which recognize specific receptors on the surface of other cells, or on various types of biotic or abiotic layers (Rickard et al., 2003). One of the types of corn on cob-like microbial interaction among filamentous fungi and cocci is observed in Figure 7. Biofilm is a complex and dynamic microbial community, and has been observed in bacteria, filamentous fungi, yeasts, and protozoa. In this context, the biofilms formed by Candida spp. are the most studied. Their formation initially entails adhesion by yeast-form cells, followed by the formation of the matrix, resulting in a tridimensional structure (Kojic and Darouiche, 2004). Mature biofilms present yeast-form cells and pseudohyphae embedded in a matrix of polysaccharides, carbohydrates, proteins, and unknown components. Yeastform cells form an adhesive layer on the surface, while the pseudohyphae compose the external layer (Baillie and Douglas, 1999; Kojic and Darouiche, 2004). The microorganisms living in the biofilm form are more resistant to microbial agents and defense from the immune system. This increased resistance is a result of the combination of various factors, such as the selection of more resistant microorganisms, Microscopy Research and Technique
ENDOTRACHEAL TUBE BIOFILM AND VENTILATOR-ASSOCIATED PNEUMONIA
309
Fig. 4. SEM-generated photomicrographs of endotracheal tubes with filamentous fungi and yeast in biofilm.
Fig. 5. SEM-generated photomicrographs of endotracheal tubes with bacilli in biofilm.
physiological adaptations such as the low rate of multiplication, and the production of EPS (Chambless et al., 2006; Szymanska, 2003). Microscopy Research and Technique
The literature points to biofilm as a potential source of infection, especially of the lungs, and its occurrence appears to be related to intubation period of the
310
P.R. DE SOUZA ET AL.
Fig. 6. SEM-generated photomicrographs of endotracheal tubes with cocci in biofilm.
endotracheal tube. Biofilm on devices has been pointed to as a cause of persistent infections, and effectiveness of the therapy involves the definitive removal of the applied and/or implanted device (Hall-Stoodley et al., 2004; Inglis, 1995; Kubota et al., 2008; Schaudinn et al., 2007). The presence of an endotracheal tube significantly affects the mucociliary transport mechanism, important in the removal of secretions or strange particles. The tube also impairs the efficacy of the coughing process, because there is no closing of the glottis. Therefore, the endotracheal tube is considered a risk factor for pneumonia because it impairs the host’s defenses and permits inhaled particles to have direct access to the lower airways, and favors microbial colonization (Adair et al., 1999; Garcia, 2005; Rello et al., 2006). Some researchers, Barbier et al. (2013), Berra et al. (2008), and Feldman et al. (1999), have demonstrated the formation of biofilm on endotracheal tubes, and explain that the tube with biofilm works as a microbial reservoir, being a potential source of contamination for the lungs of the intubated patients. Gil-Perotin et al. (2012) found a direct relationship between bacterial colonization of the airways, formation of biofilm, and development of VAP as well as implication of survival of bacteria on biofilms on endotracheal tubes of patients under mechanical ventilation.
In this study, the association between intubation period of the endotracheal tube up to 7 days or 8 days with VAP is demonstrated in Table 2. VAP has a complex and multifactorial etiology, highlighting the clinical situation of the patient, type of material and composition of the endotracheal tube, intubation technique, frequency of manipulation of the system, and insertion time of the tube, among other factors (Tablan et al., 2004). In the past few years, the scientific literature signaled the association of the endotracheal tube on the pathogenesis of VAP. The presence of this device in the airway, although necessary for the realization of invasive mechanical ventilation, has contributed to the development of this pneumonia. In this context, VAP is the lung infection most related to the presence of the endotracheal tube rather than mechanical ventilation. Thus, the name could be changed from VAP to endotracheal tube-associated pneumonia (Pneumatikos et al., 2009). Some substances have been investigated to change the properties of the endotracheal tube surface, and reduce microbial colonization. Recognition of the possibility of colonization associated with risk of infection has led to research on tubes impregnated with antimicrobials (Berra et al., 2008; Gil-Perotin et al., 2012). Other speculations were extracted in the research: one must consider the possibility of reaction by the immune system on the in vitro development of the Microscopy Research and Technique
ENDOTRACHEAL TUBE BIOFILM AND VENTILATOR-ASSOCIATED PNEUMONIA
311
Fig. 7. SEM-generated photomicrographs of endotracheal tubes with corn on cob-like microbial interaction between filamentous fungi and cocci.
TABLE 2.
Association between intubation period of endotracheal tube with the occurrence of VAP VAP Yes
No
Intubation period (days)
n
%
n
%
RR
IC
RC
IC
P
8 Up to 7
14 3
82.4 11.1
3 24
17.6 88.9
7.41
2.49–22.03
37.33
6.61–210.73
Endotracheal Tube Biofilm and Ventilator-Associated Pneumonia With Mechanical Ventilation PAULA REGINA DE SOUZA,1 DENISE DE ANDRADE,2 DANIELLE BEZERRA CABRAL,2 AND EVANDRO WATANABE3* 1
Department of Nursing, University of Brasılia, Brasılia, Distrito Federal, Brazil Department of General and Specialized Nursing, University of S~ ao Paulo at Ribeir~ ao Preto College of Nursing, Ribeir~ ao Preto, S~ ao Paulo, Brazil 3 Department of Restorative Dentistry, University of S~ ao Paulo, School of Dentistry of Riber~ ao Preto, Ribeir~ ao Preto, S~ ao Paulo, Brazil 2
KEY WORDS
biofilms; pneumonia; ventilator-associated; intubation; intratracheal; crossinfection
ABSTRACT Objective: To analyze biofilm on internal and external surfaces of endotracheal tubes after their use in critical care patients, and to produce evidence of association between use of the tube, presence of biofilm, and the occurrence of pneumonia. Methods: This was a clinical study performed at the Intensive Care Unit of an emergency hospital in the interior of S~ ao Paulo state, Brazil. Data collection involved 30 endotracheal tubes used on adult patients for a period of 48 h of mechanical ventilation for scanning electron microscopy. Results: Analysis of the biofilm on the 30 tubes by scanning electron microscopy showed various abiotic and biotic structures, predominately on the internal surface, such as: fibrin network, erythrocytes, leukocytes, cocci, bacilli, and molds, among others. The intubation period of the endotracheal tube for 8 days represented one of the risk factors for ventilator-associated pneumonia (RR 7.41, P < 0.001). Conclusions: The presence of the endotracheal tube permits microbial colonization, overall contributing to the development of biofilm and the occurrence of pneumonia. Microsc. Res. Tech. 77:305–312, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Ventilator-associated pneumonia (VAP) is the most frequent hospital infection in intensive care units (ICUs), resulting in prolonged length of stay, higher rates of morbimortality, and a significant increase of costs (Bekaert et al., 2011; Melsen et al., 2011; Nair and Niederman, 2013). Patients who undergo mechanical ventilation have a risk of 10–65% of developing pneumonia, and 24–76% risk of mortality, depending on the research method and sample as well as the therapy adopted (Augustyn, 2007; Barbier et al., 2013). The diagnosis of pneumonia should be confirmed starting at 48 h of mechanical ventilation (Masterton et al., 2007; Mietto et al., 2013; Tablan et al., 2004). The presence of the endotracheal tube is an important causal factor in the genesis of VAP, because it modifies the anatomy of the larynx, causing damages to the mucociliary system, and acting as an entry point for microorganisms to the lower airways, through microbial adherence and multiplication on the tracheal surface (biofilm) (Pinciroli et al., 2013). Biofilm is considered the new paradigm of infection, especially associated on implants of prostheses, tubes, or catheters, for example, the endotracheal tube. The presence of the endotracheal tube undoubtedly favors microbial colonization, contributing overall to the development of biofilm, which may explain the occurrence of pneumonia (Hall-Stoodley et al., 2004; Kubota et al., 2008; Schaudinn et al., 2007). Biofilm is a microbial complex, embedded in a matrix of extracellular polymeric substances (EPS), designed on C V
2014 WILEY PERIODICALS, INC.
various surface types, whether abiotic such as rocks, metals and polymers, or biotic such as vegetable and animal tissues. Further, the formation of microenvironments on the biofilm offers protection to this microbial community against biological, physical, and chemical agents from the environment (Costerton et al., 1995; Maddula et al., 2006; Santos et al., 2011). In general, studies support the theory that the formation of biofilm can be an important mechanism of tube obstruction, in addition to representing a potential source of lung infection. Added to this, studies suggest that there is a relation between the use time of the tube and production of respiratory secretions, primarily in the subglottic region (Bassetti et al., 2012; Ramirez et al., 2007; Smulders et al., 2002). Studies have suggested using scanning electron microscopy (SEM) to analyze the biofilm, whose image provides unique advantages such as high resolution and large depth of field, which allows a detailed description of the topography of biofilms and their *Correspondence to: Evandro Watanabe, Av. do Cafe, s/no, Monte Alegre, Ribeir~ ao Preto, S~ ao Paulo, Brazil. E-mail: [email protected] Received 10 September 2013; accepted in revised form 24 January 2014 REVIEW EDITOR: Prof. George Perry Part of doctoral thesis, titled “Microbiological molecular-genetic analysis of the orotracheal biota of critical care patients: supports in the prevention of ventilator-associated pneumonia.” Contract grant sponsor: Brazilian funding agencies FAPESP and CAPES (Ph.D. fellowship). DOI 10.1002/jemt.22344 Published online 11 February 2014 in Wiley Online Library (wileyonlinelibrary. com).
306
P.R. DE SOUZA ET AL.
Fig. 1. SEM-generated photomicrographs of the internal (left side) and external (right side) surfaces of the sterilized endotracheal tube without the presence of biofilm.
TABLE 1. Morphological characteristics of biofilm on the 30 endotracheal tubes (internal and external surfaces) used on patients submitted to mechanical ventilation Internal surface Characteristics EPS matrix Compacted Fragmented Honeycomb Erythrocytes Fibrin network Leukocytes Cocci Microbial interaction Mold Bacilli
Yes No Yes No Yes No Yes No Yes No Yes No Yes No
External surface
n
%
n
%
22 4 4 3 7 5 25 5 25 1 29 1 29 1 2 0 30
73.3 13.3 13.3 10.0 90.0 16.7 83.3 16.7 83.3 3.3 96.7 3.3 96.7 3.3 96.7 0 100
12 17 1 2 28 1 29 2 28 0 30 0 30 0 30 1 29
40.0 56.7 3.3 6.7 93.3 3.3 96.7 6.7 93.3 0 100 0 100 0 100 3.3 96.7
n 5 number of samples; EPS 5 extracellular polymeric substances.
structure (Baldasso et al., 2012; Inglis, 1989, 1995; Schaudinn et al., 2007; Sottile et al., 1986). The aim of this study was to analyze the biofilm on internal and external surfaces of endotracheal tubes after their use on critical care patients, and to produce evidence of the association between use of the tube, presence of biofilm, and the occurrence of pneumonia. MATERIALS AND METHODS This study had a clinical focus, and was performed at the adult ICU of a public teaching hospital in the interior of the state of S~ ao Paulo, Brazil. The research was approved by the Research Ethics Committee (protocol 987/2008) of the studied hospital, and involved 30 study subjects of both sexes, aged between 18 and 60 years, who were critical care patients with endotracheal tubes who underwent mechanical ventilation for a period of 48 h of tracheal
intubation. Patients who developed pneumonia within 48 h of admission to the ICU as well as those with accidental extubation and intubation at another hospital were excluded from this study. Following extubation by physician recommendation, the endotracheal tubes were placed in sterilized, separate plastic bags, in a class II biological safety cabinet, for preparation of the samples and later analysis by SEM (Silveira, 2007). Sample preparation consisted of the process of dehydration with ethyl alcohol in different concentrations of 30, 50, 70, 90, and 95%, sequentially, for a period of 10 min, and 100%, three times, for 20 min. After dehydration, proceeded drying with CO2 (BalTec CPD 030, Critical Point Dryer—Furstentem—Liechtenstein), mounting on metal supports with a conductive adhesive (Colloidal Graphite Isopropanol Base), and vacuum metallization in gold for analysis of the surfaces of the endotracheal tubes by SEM. In this research, 30 fragments of endotracheal tubes (internal and external surfaces) used on critical care patients were evaluated, totaling 112 SEM-generated photographic records. In this research, biofilms were analyzed by scanning electron microscope (SEM) and a sterilized endotracheal tube was used as negative control. Moreover, all molecules and microorganisms were morphologically identified and EPS matrix structures were observed and classified as described by Donlan and Costerton (2002) and Schaudinn et al. (2007). The physician diagnosed cases of VAP based on clinical and radiological examination in accordance with CDC criteria (Tablan et al., 2004). Analysis of the association between potential predictors (endotracheal tube intubation period and outcome (ventilator-associated pneumonia) and agreement between the different measurements was performed through bivariate contingency tables. RESULTS AND DISCUSSION To evaluate the presence of biofilm on the samples, an unused, sterilized endotracheal tube was used as a negative control, which presented irregularities on the Microscopy Research and Technique
ENDOTRACHEAL TUBE BIOFILM AND VENTILATOR-ASSOCIATED PNEUMONIA
307
Fig. 2. SEM-generated photomicrographs of the endotracheal tubes with biofilm, compact EPS matrix (up side) and fragmented EPS matrix (down side).
internal and external surfaces, characteristics of the material that can favor the development of biofilm, as observed in Figure 1. All the samples presented biofilm, and the morphological characteristics are shown on Table 1. According to Table 1, the presence of the compacted and fragmented EPS matrix is more evident on the internal and external surfaces of the endotracheal tubes, respectively, as the internal surfaces of the tubes show less mechanical friction than the external, in direct contact with the trachea of the patient (Fig. 2). It was observed that the biofilm on the distal surfaces (internal and external) of the endotracheal tubes had diversified structures, not only in terms of morphological characteristics of the microbiota but also in the composition of elements such as fibrin, erythrocytes, and leukocytes, as shown in Table 1. In other words, it should also be considered that there is a direct relationship between use time of devices and the formation of biofilm, and that the use time represents an important factor in the occurrence of infection. It is important to note that the presence of biofilm on the endotracheal tubes of critical care patients, independent of the occurrence or not of VAP, reinforces the data from the literature that microorganisms Microscopy Research and Technique
adhere to the biomaterials, forming a microbial community on these surfaces (Berra et al., 2008; Chambless et al., 2006; Costerton et al., 1995; Hall-Stoodley et al., 2004). The EPS produced by microorganisms are constituted of different substances, intermediate microbial adherence to the surface of the material, and constitute the biofilm matrix. This matrix corresponds to approximately 85% of the volume of the biofilm (Donlan and Costerton, 2002). Furthermore, a structure resembling a “honeycomb,” documented in Figure 3 and described by Schaudinn et al. (2007), was clear on the external and internal surfaces of three endotracheal tubes. The honeycomb structure was observed in biofilm produced by Staphylococcus epidermidis and Pseudomonas spp., showing a tridimensional aspect with six elastic, symmetrical axes that confer flexibility and stability, permitting deformity to occur in response to stress, and subsequent return to the original shape. This characteristic endows the biofilm with greater resistance to adversity from the environment as well as antimicrobial action, considered an important virulence factor. It also favors multidirectional flow throughout the extension of the biofilm, allowing for less energy expense, and greater area of nutrient absorption (Schaudinn et al., 2007).
308
P.R. DE SOUZA ET AL.
Fig. 3. SEM-generated photomicrographs of endotracheal tubes with biofilm in the shape of a “honeycomb.”
Considering that the biofilm is constituted of approximately 90–95% water, during drying (critical point), a preparation stage of the sample for SEM, a substantial proportion of the volume of this biofilm is eliminated. This dehydration provokes the destruction of the structural architecture of the biofilm. Thus, the SEM tends to emphasize the microorganisms, sometimes observed in openings or fissures in the biofilm (Inglis, 1995; Schaudinn et al., 2007; Sottile et al., 1986). Thus, a concerning result was the proven presence of filamentous fungi and yeast (26.6%), bacilli (13.3%), cocci (6.6%), and microbial interactions (6.6%) on the endotracheal tubes (Table 1, Figs. 4–7). The bacteria commonly isolated from endotracheal tube biofilms include: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus intermedius, Streptococcus viridans, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Added to these are the following species of coagulasenegative Staphylococcus: Staphylococcus capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus auricularis, and Staphylococcus carnosus (Barbier et al., 2013; Feldman, 1999; Kojic and Darouiche, 2004; Otto, 2008). The microbial interactions that permit the coaggregation of different species, or even microbial
genera in a biofilm, generally involve the participation of adhesins (adherence molecules present in fimbriae or dispersed along the cell surface), which recognize specific receptors on the surface of other cells, or on various types of biotic or abiotic layers (Rickard et al., 2003). One of the types of corn on cob-like microbial interaction among filamentous fungi and cocci is observed in Figure 7. Biofilm is a complex and dynamic microbial community, and has been observed in bacteria, filamentous fungi, yeasts, and protozoa. In this context, the biofilms formed by Candida spp. are the most studied. Their formation initially entails adhesion by yeast-form cells, followed by the formation of the matrix, resulting in a tridimensional structure (Kojic and Darouiche, 2004). Mature biofilms present yeast-form cells and pseudohyphae embedded in a matrix of polysaccharides, carbohydrates, proteins, and unknown components. Yeastform cells form an adhesive layer on the surface, while the pseudohyphae compose the external layer (Baillie and Douglas, 1999; Kojic and Darouiche, 2004). The microorganisms living in the biofilm form are more resistant to microbial agents and defense from the immune system. This increased resistance is a result of the combination of various factors, such as the selection of more resistant microorganisms, Microscopy Research and Technique
ENDOTRACHEAL TUBE BIOFILM AND VENTILATOR-ASSOCIATED PNEUMONIA
309
Fig. 4. SEM-generated photomicrographs of endotracheal tubes with filamentous fungi and yeast in biofilm.
Fig. 5. SEM-generated photomicrographs of endotracheal tubes with bacilli in biofilm.
physiological adaptations such as the low rate of multiplication, and the production of EPS (Chambless et al., 2006; Szymanska, 2003). Microscopy Research and Technique
The literature points to biofilm as a potential source of infection, especially of the lungs, and its occurrence appears to be related to intubation period of the
310
P.R. DE SOUZA ET AL.
Fig. 6. SEM-generated photomicrographs of endotracheal tubes with cocci in biofilm.
endotracheal tube. Biofilm on devices has been pointed to as a cause of persistent infections, and effectiveness of the therapy involves the definitive removal of the applied and/or implanted device (Hall-Stoodley et al., 2004; Inglis, 1995; Kubota et al., 2008; Schaudinn et al., 2007). The presence of an endotracheal tube significantly affects the mucociliary transport mechanism, important in the removal of secretions or strange particles. The tube also impairs the efficacy of the coughing process, because there is no closing of the glottis. Therefore, the endotracheal tube is considered a risk factor for pneumonia because it impairs the host’s defenses and permits inhaled particles to have direct access to the lower airways, and favors microbial colonization (Adair et al., 1999; Garcia, 2005; Rello et al., 2006). Some researchers, Barbier et al. (2013), Berra et al. (2008), and Feldman et al. (1999), have demonstrated the formation of biofilm on endotracheal tubes, and explain that the tube with biofilm works as a microbial reservoir, being a potential source of contamination for the lungs of the intubated patients. Gil-Perotin et al. (2012) found a direct relationship between bacterial colonization of the airways, formation of biofilm, and development of VAP as well as implication of survival of bacteria on biofilms on endotracheal tubes of patients under mechanical ventilation.
In this study, the association between intubation period of the endotracheal tube up to 7 days or 8 days with VAP is demonstrated in Table 2. VAP has a complex and multifactorial etiology, highlighting the clinical situation of the patient, type of material and composition of the endotracheal tube, intubation technique, frequency of manipulation of the system, and insertion time of the tube, among other factors (Tablan et al., 2004). In the past few years, the scientific literature signaled the association of the endotracheal tube on the pathogenesis of VAP. The presence of this device in the airway, although necessary for the realization of invasive mechanical ventilation, has contributed to the development of this pneumonia. In this context, VAP is the lung infection most related to the presence of the endotracheal tube rather than mechanical ventilation. Thus, the name could be changed from VAP to endotracheal tube-associated pneumonia (Pneumatikos et al., 2009). Some substances have been investigated to change the properties of the endotracheal tube surface, and reduce microbial colonization. Recognition of the possibility of colonization associated with risk of infection has led to research on tubes impregnated with antimicrobials (Berra et al., 2008; Gil-Perotin et al., 2012). Other speculations were extracted in the research: one must consider the possibility of reaction by the immune system on the in vitro development of the Microscopy Research and Technique
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Fig. 7. SEM-generated photomicrographs of endotracheal tubes with corn on cob-like microbial interaction between filamentous fungi and cocci.
TABLE 2.
Association between intubation period of endotracheal tube with the occurrence of VAP VAP Yes
No
Intubation period (days)
n
%
n
%
RR
IC
RC
IC
P
8 Up to 7
14 3
82.4 11.1
3 24
17.6 88.9
7.41
2.49–22.03
37.33
6.61–210.73
