Journal of Hospital Infection 90 (2015) 347e350 Available online at www.sciencedirect.com
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Short report
Survival of vancomycin-intermediate Staphylococcus aureus on hospital surfaces M.N. Zarpellon a, b, A.C. Gales c, A.L. Sasaki a, G.J. Selhorst a, T.C. Menegucci a, C.L. Cardoso a, L.B. Garcia a, M.C.B. Tognim a, * a
Departamento de Cieˆncias Ba´sicas da Sau´de, Universidade Estadual de Maringa´, PR, Brazil Hospital Universita´rio de Maringa´, Universidade Estadual de Maringa´, PR, Brazil c Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil b
A R T I C L E
I N F O
Article history: Received 29 November 2014 Accepted 6 April 2015 Available online 30 April 2015 Keywords: Staphylococcus aureus Vancomycin Survival Biofilm Hydrophobicity Adherence
S U M M A R Y
Background: Contaminated surfaces play an important role in the transmission of certain pathogens that are responsible for healthcare-associated infections. Although previous studies have shown that meticillin-resistant Staphylococcus aureus (MRSA) can survive on dry surfaces at room temperature, no published data regarding vancomycin-intermediate S. aureus (VISA) are available to date. Aim: To compare the survival time on different types of surfaces, cell-surface hydrophobicity, adherence to abiotic surfaces and biofilm formation of meticillin-susceptible S. aureus (MSSA), MRSA and VISA. Methods: Survival of the S. aureus strains was tested on latex, cotton fabric, vinyl flooring and formica. Cell-surface hydrophobicity was determined using the hydrocarbon interaction affinity method. Adhesion to abiotic surfaces was tested on granite, latex (gloves), glass, vinyl flooring and formica. Biofilm formation was evaluated at 6, 12, 24 and 48 h. Findings: All of the samples survived on the vinyl flooring and formica for at least 40 days. VISA survived on both surfaces for more than 45 days. All of the strains were highly hydrophobic. VISA adhered to latex, vinyl flooring and formica. Biofilm formation increased for all of the tested strains within 6e24 h. Conclusion: VISA present high survival, adherence and cell-surface hydrophobicity. Therefore, as the treatment of patients with VISA is a significant challenge for clinicians, greater care with cleaning and disinfection of different types of surfaces in healthcare facilities is recommended because these may become important reservoirs of multiresistant pathogens. ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Address: Laborato ´rio de Microbiologia, Departamento de Cie ˆncias Ba ´sicas da Sau ´de, Universidade Estadual de Maringa ´, Avenida Colombo 5790, Maringa ´, Parana ´, CEP 87020-900, Brazil. Tel.: þ55 44 3011 4952; fax: þ55 44 3011-5941. E-mail address: [email protected] (M.C.B. Tognim).
Since the first strain of vancomycin-intermediate Staphylococcus aureus (VISA) was described in Japan,1 several other isolates have been reported worldwide.2 VISA have thick cell walls, and may also have enhanced ability to form a thick biofilm.1e4
http://dx.doi.org/10.1016/j.jhin.2015.04.005 0195-6701/ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
348
M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
Bacterial biofilms are difficult to remove because of their increased resistance to detergents and disinfectants.5 The presence of multi-drug-resistant bacteria, including meticillinresistant S. aureus (MRSA), that are protected within biofilms may represent an important mechanism for promoting the prolonged permanence of pathogens in hospital environments.4e6 Contacts of healthcare personnel and patients with MRSA-contaminated environments may result in the transmission of, or infection by, these pathogens.5e8 Although previous studies have shown that MRSA can survive on dry surfaces at room temperature, no published data regarding VISA are available to date. Thus, the aim of this study was to compare the survival time on different types of surfaces, cell-surface hydrophobicity, adherence to abiotic surfaces and biofilm formation of meticillin-susceptible S. aureus (MSSA), MRSA and VISA.
Methods Test organisms Three different strains of S. aureus were tested in this study: ATCC 25923 (MSSA), ATCC 33591 (MRSA) and VISA, isolated from the blood culture of a patient with endocarditis who was treated with vancomycin.3
VISA The minimum inhibitory concentration of the original VISA sample was 8 mg/mL, which was induced to express thickening of the cell wall. Vancomycin resistance was induced as described previously.3
Surface survival Test bodies Survival of the three S. aureus strains was tested on latex (gloves), 100% cotton fabric, vinyl flooring and formica. Initially, test bodies (TBs) that measured 10 20 mm were washed and conditioned in flasks with distilled water (latex, formica and vinyl flooring) and without water (cotton fabric), and sterilized in an autoclave for 15 min at 121 C. Next, the TBs were transferred aseptically to 90 15 mm sterile Petri dishes and dried in an oven at 50 C. Plates that contained approximately 20 TBs each were maintained in a biosafety hood throughout the experiment. Artificial contamination Ten-microlitre volumes of an 18e24-h nutrient broth culture of each test micro-organism, adjusted to achieve 0.5 MacFarland standard turbidity-equivalent (108 UFC/mL), were applied to the TB surfaces. Recovery of test micro-organisms After the inoculum dried, the TBs were transferred to sterile 13 100 mm test tubes and kept in the laboratory for 45 days. The micro-organisms that survived drying were recovered daily in duplicate, transferred aseptically to tubes that contained 3 mL of brain heart infusion broth, and vortexed for 1 min. The tubes were incubated at 37 C and observed daily for six days. Turbidity of the tubes was considered to be positive if the characteristics of the developed organisms were compatible
with S. aureus. The temperature and relative humidity of the environment were determined daily.
Cell-surface hydrophobicity The cell-surface hydrophobicity of the test micro-organisms was determined using the hydrocarbon interaction affinity method and 1 mL of xylene in accordance with Teixeira et al.9 The hydrophobicity index (HI) was calculated using the following formula: HI ¼ (A660 B660) 100/A660, where A660 is the optical density (OD) of the microbial suspension before xylene treatment, and B660 is the OD of the aqueous phase obtained after xylene treatment.9,10 Additionally, five isolates of each bacterial group (MSSA, MRSA and VISA) were tested for hydrophobicity. Clinical isolates from different times were selected for MSSA and MRSA, while Mu3 and Mu50 strains and three clinical samples obtained from medical centres located in different areas of Brazil were used for VISA. The VISA isolates selected were cultured in dishes containing Mueller Hinton agar, with vancomycin added before the hydrophobicity test was performed. These experiments were performed in triplicate and repeated in two independent sets of experiments.
Adherence to abiotic surfaces Adhesion to abiotic surfaces was tested using granite, latex (gloves), glass, vinyl flooring and formica, as described previously.11
Biofilm formation The biofilm formation and OD of planktonic cells were evaluated at 6, 12, 24 and 48 h of aerobic incubation at 37 C in microtitre-plate systems, as described previously.12
Results Surface survival All of the samples survived on the vinyl flooring and formica for at least 40 days. VISA survived on both surfaces for more than 45 days, which was the maximum time of experimental observation. On latex, VISA and MSSA survived for one day and MRSA survived for two days. On cotton fabric, MRSA and MSSA survived for 18 days and VISA survived for three days. The mean temperature and relative humidity of the air during the experiments were 30 C (range 22e38 C) and 60% (range 39e79%), respectively (Table I).
Cell-surface hydrophobicity All three test strains and the additional isolates tested were highly hydrophobic (HI >50%). HI values are show in Table I.
Adherence to abiotic surfaces MSSA adhered to latex and glass. VISA adhered to latex, vinyl flooring and formica, and MRSA adhered to latex, glass, vinyl flooring and formica (Table I).
M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
VISA 1 3 >45 >45 HI (%) 87.62 89.83 91.15 95.43 98.55 97.32 87.34 95.48 87.75 97.85 90.52 86.85 89.57 72.84 87.97 97.50 91.18 88.30 VISA // þ/þ/ // þ/þ/þ þ/þ/þ
OD, optical density; HI, hydrophobicity index; , no adherence; þ, adherence. a Average of two independent sets of experiments performed in triplicate.
Biofilm formation In aerobic incubation at 37 C, biofilm formation increased for all three test strains within 6e24 h. MRSA were the largest biofilm producers, with an increase in OD that could be detected until 48 h (OD6h ¼ 0.029, OD48h ¼ 0.881). For VISA, biofilm formation at 24 h was 10 times greater than biofilm formation at 6 h (OD6h ¼ 0.023, OD24h ¼ 0.225). Among the test strains, MSSA produced the least biofilm (Figure 1). Planktonic cells decreased over time for all tested strains (Figure 1).
Discussion Contaminated surfaces play an important role in the transmission of certain pathogens that are responsible for
Optical density
Duration of survival (days) Fomite MSSA MRSA Latex 1 2 Cotton fabric 18 18 Vinyl flooring >45 41 Formica 43 40 Cell-surface hydrophobicitya Isolate/source Initial OD Final OD MSSA ATCC 25923 0.404 0.050 Sa196 0.413 0.042 Sa225 0.407 0.036 Sa278 0.416 0.019 Sa289 0.414 0.006 Sa296 0.410 0.011 MRSA ATCC 33591 0.379 0.048 Sa158 0.376 0.017 Sa226 0.408 0.050 Sa329 0.418 0.009 Sa382 0.401 0.038 Sa413 0.403 0.053 VISA VISA 0.374 0.039 Mu50 0.416 0.113 Mu3 0.399 0.048 62904 0.400 0.010 SP clone 0.397 0.035 SS clone 0.393 0.046 Adherence to abiotic surfaces (Day 1/2/3) Fomite MSSA MRSA Granite // // Latex þ// þ/þ/þ Glass þ// þ// Vinyl flooring // //þ Formica // þ//þ
a
b Optical density
Table I Meticillin-susceptible Staphylococcus aureus (MSSA), meticillinresistant S. aureus (MRSA) and vancomycin-intermediate S. aureus (VISA): duration of survival, hydrophobicity and adherence on different fomites
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
6
12
349
24
48
Time (h) 1.2 1 0.8 0.6 0.4 0.2 0
6
12
24
48
Time (h)
Figure 1. Biofilm formation (a) and planktonic cell determination (b) at different times. Green line, meticillin-susceptible Staphylococcus aureus; red line, meticillin-resistant S. aureus; blue line, vancomycin-intermediate S. aureus.
healthcare-associated infections, such as MRSA, which can survive on dry surfaces for months.6e8 In the present study, MSSA, MRSA and VISA survived for at least 40 days on rough (vinyl flooring) and smooth (formica) surfaces. These findings agree, in part, with a previous study by Huang et al., whose results indicated less survival on smooth surfaces.8 Notably, in the present study, VISA survived for the entire experimental period (45 days) on both formica and vinyl flooring, whereas MSSA only demonstrated a comparable survival time on vinyl flooring (Table I). Thus, this study highlights the ease with which these strains, especially VISA, can survive in hospital environments, which could result in infections and even outbreaks. However, it is important to point out that different temperature and humidity conditions may result in important variations in the survival profile of S. aureus on dry surfaces.6 Taking into consideration the fact that the present study was conducted at high humidity (39e79%) and temperature (22e38 C), the findings may be readily applicable in countries with similar climatic characteristics, but not in colder and drier regions of the world such as northern Europe. Hydrophobic interactions play an important role in the nonspecific adherence of micro-organisms to biotic and abiotic surfaces.11 Thus, high bacterial hydrophobicity may favour the colonization, permanence and dissemination of pathogens in hospital environments.10,11 HI may vary between strains; therefore, five isolates of each bacteria (MSSA, MRSA and VISA) were also included in the hydrophobicity tests. All three test strains and the additional isolates tested were highly hydrophobic (HI >50%) (Table I). The results for VISA are particularly interesting as, to the authors’ knowledge, no data have been reported previously.
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M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
The high hydrophobicity of VISA seems to be associated with its high surface adherence to vinyl flooring and formica, and the survival pattern observed. Under hot and humid conditions, cotton fabric would tend to absorb moisture, and this may explain why VISA (HI >89.57%) did not survive for long on this material. On the other hand, MSSA and MRSA also showed high HI but with different patterns of adherence to abiotic surfaces. In this study, the results from the hydrophobicity tests could not be related to the other parameters assessed (survival, adherence to abiotic surfaces and biofilm production). No adherence was observed on granite and little adherence was observed on glass, indicating that these types of materials could be considered for furniture or other surfaces in hospital settings. Due to the shortage of studies on the adherence of bacteria such as VISA to the materials used in hospital surfaces, further research is necessary to fully understand why some surfaces are much less favourable to VISA than others. Surface adherence is the preliminary factor for some microorganisms to form the complex extracellular matrix of polymeric substances that constitute biofilms.4,10,11 Biofilmforming bacteria may resist death by antibiotics, biocides and disinfectants compared with planktonic cells.5,10,12 The present study found that MRSA produced more biofilm compared with the other two test strains (Figure 1). These data were obtained on a polystyrene surface at 37 C with aeration, and may not be generalizable to other conditions, as the amount of biofilm formed by the different strains may vary between surfaces and environmental conditions.5,10 To the best of the authors’ knowledge, this is the first study to demonstrate that VISA presents high survival, adherence and cell-surface hydrophobicity. Therefore, as the treatment of patients with VISA is a significant challenge for clinicians, greater care with cleaning and disinfection of different types of surfaces in healthcare facilities is recommended as these may become important reservoirs of multi-resistant pathogens.
Acknowledgements The authors wish to thank Antonio Carlos Correa and Michael Arends for English text review. VISA were kindly provided by Ana Cristina Gales and Alessandro Conrado de Oliveira Silveira. Conflict of interest statement None declared.
Funding source This study was supported by the Coordenac ¸a ˜o de Aperfei¸ coamento de Pessoal de Nı´vel Superior and Fundac ¸a ˜o Arauca ´ria.
References 1. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 1997;40:135e136. 2. Howden BP, Peleg AY, Stinear TP. The evolution of vancomycin intermediate Staphylococcus aureus (VISA): an heterogenous VISA. Infect Genet Evol 2014;21:575e582. 3. Andrade-Baiocchi S, Tognim MC, Baiocchi OC, Sader HS. Endocarditis due to glycopeptide-intermediate Staphylococcus aureus: case report and strain characterization. Diagn Microbiol Infect Dis 2003;45:149e152. 4. Sakoulas G, Moellering Jr RC, Eliopoulos GM. Adaptation of methicillin-resistant Staphylococcus aureus in the face of vancomycin therapy. Clin Infect Dis 2006;42(Suppl. 1):S40eS50. 5. Vickery K, Deva A, Jacombs A, Allan J, Valente P, Gosbell IB. Presence of biofilm containing viable multiresistant organisms despite terminal cleaning on clinical surfaces in an intensive care unit. J Hosp Infect 2012;80:52e55. 6. Wagenvoort JHT, Sluijsmans W, Penders RJR. Better environmental survival of outbreak vs. sporadic MRSA isolates. J Hosp Infect 2000;45:231e234. 7. Weber DJ, Rutala WA. Understanding and preventing transmission of healthcare-associated pathogens due to the contaminated hospital environment. Infect Control Hosp Epidemiol 2013;34:449e452. 8. Huang R, Mehta S, Weed D, Price CS. Methicillin-resistant Staphylococcus aureus survival on hospital fomites. Infect Control Hosp Epidemiol 2006;27:1267e1269. 9. Teixeira LA, Figueiredo AMS, Ferreira BT, et al. Sialic acid content and surface hydrophobicity of group B streptococci. Epidemiol Infect 1993;110:87e94. 10. Di Ciccio P, Vergara A, Festino AR, et al. Biofilm formation by Staphylococcus aureus on food contact surfaces: relationship with temperature and cell surface hydrophobicity. Food Control 2015;50:930e936. 11. Rosenberg M. Bacterial adherence to polystyrene: a replica method of screening for bacterial hydrophobicity. Appl Environ Microbiol 1981;42:375e377. 12. Pitts B, Hamilton MA, Zelver N, Stewart PS. A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 2003;54:269e276.
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Short report
Survival of vancomycin-intermediate Staphylococcus aureus on hospital surfaces M.N. Zarpellon a, b, A.C. Gales c, A.L. Sasaki a, G.J. Selhorst a, T.C. Menegucci a, C.L. Cardoso a, L.B. Garcia a, M.C.B. Tognim a, * a
Departamento de Cieˆncias Ba´sicas da Sau´de, Universidade Estadual de Maringa´, PR, Brazil Hospital Universita´rio de Maringa´, Universidade Estadual de Maringa´, PR, Brazil c Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil b
A R T I C L E
I N F O
Article history: Received 29 November 2014 Accepted 6 April 2015 Available online 30 April 2015 Keywords: Staphylococcus aureus Vancomycin Survival Biofilm Hydrophobicity Adherence
S U M M A R Y
Background: Contaminated surfaces play an important role in the transmission of certain pathogens that are responsible for healthcare-associated infections. Although previous studies have shown that meticillin-resistant Staphylococcus aureus (MRSA) can survive on dry surfaces at room temperature, no published data regarding vancomycin-intermediate S. aureus (VISA) are available to date. Aim: To compare the survival time on different types of surfaces, cell-surface hydrophobicity, adherence to abiotic surfaces and biofilm formation of meticillin-susceptible S. aureus (MSSA), MRSA and VISA. Methods: Survival of the S. aureus strains was tested on latex, cotton fabric, vinyl flooring and formica. Cell-surface hydrophobicity was determined using the hydrocarbon interaction affinity method. Adhesion to abiotic surfaces was tested on granite, latex (gloves), glass, vinyl flooring and formica. Biofilm formation was evaluated at 6, 12, 24 and 48 h. Findings: All of the samples survived on the vinyl flooring and formica for at least 40 days. VISA survived on both surfaces for more than 45 days. All of the strains were highly hydrophobic. VISA adhered to latex, vinyl flooring and formica. Biofilm formation increased for all of the tested strains within 6e24 h. Conclusion: VISA present high survival, adherence and cell-surface hydrophobicity. Therefore, as the treatment of patients with VISA is a significant challenge for clinicians, greater care with cleaning and disinfection of different types of surfaces in healthcare facilities is recommended because these may become important reservoirs of multiresistant pathogens. ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Address: Laborato ´rio de Microbiologia, Departamento de Cie ˆncias Ba ´sicas da Sau ´de, Universidade Estadual de Maringa ´, Avenida Colombo 5790, Maringa ´, Parana ´, CEP 87020-900, Brazil. Tel.: þ55 44 3011 4952; fax: þ55 44 3011-5941. E-mail address: [email protected] (M.C.B. Tognim).
Since the first strain of vancomycin-intermediate Staphylococcus aureus (VISA) was described in Japan,1 several other isolates have been reported worldwide.2 VISA have thick cell walls, and may also have enhanced ability to form a thick biofilm.1e4
http://dx.doi.org/10.1016/j.jhin.2015.04.005 0195-6701/ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
348
M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
Bacterial biofilms are difficult to remove because of their increased resistance to detergents and disinfectants.5 The presence of multi-drug-resistant bacteria, including meticillinresistant S. aureus (MRSA), that are protected within biofilms may represent an important mechanism for promoting the prolonged permanence of pathogens in hospital environments.4e6 Contacts of healthcare personnel and patients with MRSA-contaminated environments may result in the transmission of, or infection by, these pathogens.5e8 Although previous studies have shown that MRSA can survive on dry surfaces at room temperature, no published data regarding VISA are available to date. Thus, the aim of this study was to compare the survival time on different types of surfaces, cell-surface hydrophobicity, adherence to abiotic surfaces and biofilm formation of meticillin-susceptible S. aureus (MSSA), MRSA and VISA.
Methods Test organisms Three different strains of S. aureus were tested in this study: ATCC 25923 (MSSA), ATCC 33591 (MRSA) and VISA, isolated from the blood culture of a patient with endocarditis who was treated with vancomycin.3
VISA The minimum inhibitory concentration of the original VISA sample was 8 mg/mL, which was induced to express thickening of the cell wall. Vancomycin resistance was induced as described previously.3
Surface survival Test bodies Survival of the three S. aureus strains was tested on latex (gloves), 100% cotton fabric, vinyl flooring and formica. Initially, test bodies (TBs) that measured 10 20 mm were washed and conditioned in flasks with distilled water (latex, formica and vinyl flooring) and without water (cotton fabric), and sterilized in an autoclave for 15 min at 121 C. Next, the TBs were transferred aseptically to 90 15 mm sterile Petri dishes and dried in an oven at 50 C. Plates that contained approximately 20 TBs each were maintained in a biosafety hood throughout the experiment. Artificial contamination Ten-microlitre volumes of an 18e24-h nutrient broth culture of each test micro-organism, adjusted to achieve 0.5 MacFarland standard turbidity-equivalent (108 UFC/mL), were applied to the TB surfaces. Recovery of test micro-organisms After the inoculum dried, the TBs were transferred to sterile 13 100 mm test tubes and kept in the laboratory for 45 days. The micro-organisms that survived drying were recovered daily in duplicate, transferred aseptically to tubes that contained 3 mL of brain heart infusion broth, and vortexed for 1 min. The tubes were incubated at 37 C and observed daily for six days. Turbidity of the tubes was considered to be positive if the characteristics of the developed organisms were compatible
with S. aureus. The temperature and relative humidity of the environment were determined daily.
Cell-surface hydrophobicity The cell-surface hydrophobicity of the test micro-organisms was determined using the hydrocarbon interaction affinity method and 1 mL of xylene in accordance with Teixeira et al.9 The hydrophobicity index (HI) was calculated using the following formula: HI ¼ (A660 B660) 100/A660, where A660 is the optical density (OD) of the microbial suspension before xylene treatment, and B660 is the OD of the aqueous phase obtained after xylene treatment.9,10 Additionally, five isolates of each bacterial group (MSSA, MRSA and VISA) were tested for hydrophobicity. Clinical isolates from different times were selected for MSSA and MRSA, while Mu3 and Mu50 strains and three clinical samples obtained from medical centres located in different areas of Brazil were used for VISA. The VISA isolates selected were cultured in dishes containing Mueller Hinton agar, with vancomycin added before the hydrophobicity test was performed. These experiments were performed in triplicate and repeated in two independent sets of experiments.
Adherence to abiotic surfaces Adhesion to abiotic surfaces was tested using granite, latex (gloves), glass, vinyl flooring and formica, as described previously.11
Biofilm formation The biofilm formation and OD of planktonic cells were evaluated at 6, 12, 24 and 48 h of aerobic incubation at 37 C in microtitre-plate systems, as described previously.12
Results Surface survival All of the samples survived on the vinyl flooring and formica for at least 40 days. VISA survived on both surfaces for more than 45 days, which was the maximum time of experimental observation. On latex, VISA and MSSA survived for one day and MRSA survived for two days. On cotton fabric, MRSA and MSSA survived for 18 days and VISA survived for three days. The mean temperature and relative humidity of the air during the experiments were 30 C (range 22e38 C) and 60% (range 39e79%), respectively (Table I).
Cell-surface hydrophobicity All three test strains and the additional isolates tested were highly hydrophobic (HI >50%). HI values are show in Table I.
Adherence to abiotic surfaces MSSA adhered to latex and glass. VISA adhered to latex, vinyl flooring and formica, and MRSA adhered to latex, glass, vinyl flooring and formica (Table I).
M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
VISA 1 3 >45 >45 HI (%) 87.62 89.83 91.15 95.43 98.55 97.32 87.34 95.48 87.75 97.85 90.52 86.85 89.57 72.84 87.97 97.50 91.18 88.30 VISA // þ/þ/ // þ/þ/þ þ/þ/þ
OD, optical density; HI, hydrophobicity index; , no adherence; þ, adherence. a Average of two independent sets of experiments performed in triplicate.
Biofilm formation In aerobic incubation at 37 C, biofilm formation increased for all three test strains within 6e24 h. MRSA were the largest biofilm producers, with an increase in OD that could be detected until 48 h (OD6h ¼ 0.029, OD48h ¼ 0.881). For VISA, biofilm formation at 24 h was 10 times greater than biofilm formation at 6 h (OD6h ¼ 0.023, OD24h ¼ 0.225). Among the test strains, MSSA produced the least biofilm (Figure 1). Planktonic cells decreased over time for all tested strains (Figure 1).
Discussion Contaminated surfaces play an important role in the transmission of certain pathogens that are responsible for
Optical density
Duration of survival (days) Fomite MSSA MRSA Latex 1 2 Cotton fabric 18 18 Vinyl flooring >45 41 Formica 43 40 Cell-surface hydrophobicitya Isolate/source Initial OD Final OD MSSA ATCC 25923 0.404 0.050 Sa196 0.413 0.042 Sa225 0.407 0.036 Sa278 0.416 0.019 Sa289 0.414 0.006 Sa296 0.410 0.011 MRSA ATCC 33591 0.379 0.048 Sa158 0.376 0.017 Sa226 0.408 0.050 Sa329 0.418 0.009 Sa382 0.401 0.038 Sa413 0.403 0.053 VISA VISA 0.374 0.039 Mu50 0.416 0.113 Mu3 0.399 0.048 62904 0.400 0.010 SP clone 0.397 0.035 SS clone 0.393 0.046 Adherence to abiotic surfaces (Day 1/2/3) Fomite MSSA MRSA Granite // // Latex þ// þ/þ/þ Glass þ// þ// Vinyl flooring // //þ Formica // þ//þ
a
b Optical density
Table I Meticillin-susceptible Staphylococcus aureus (MSSA), meticillinresistant S. aureus (MRSA) and vancomycin-intermediate S. aureus (VISA): duration of survival, hydrophobicity and adherence on different fomites
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
6
12
349
24
48
Time (h) 1.2 1 0.8 0.6 0.4 0.2 0
6
12
24
48
Time (h)
Figure 1. Biofilm formation (a) and planktonic cell determination (b) at different times. Green line, meticillin-susceptible Staphylococcus aureus; red line, meticillin-resistant S. aureus; blue line, vancomycin-intermediate S. aureus.
healthcare-associated infections, such as MRSA, which can survive on dry surfaces for months.6e8 In the present study, MSSA, MRSA and VISA survived for at least 40 days on rough (vinyl flooring) and smooth (formica) surfaces. These findings agree, in part, with a previous study by Huang et al., whose results indicated less survival on smooth surfaces.8 Notably, in the present study, VISA survived for the entire experimental period (45 days) on both formica and vinyl flooring, whereas MSSA only demonstrated a comparable survival time on vinyl flooring (Table I). Thus, this study highlights the ease with which these strains, especially VISA, can survive in hospital environments, which could result in infections and even outbreaks. However, it is important to point out that different temperature and humidity conditions may result in important variations in the survival profile of S. aureus on dry surfaces.6 Taking into consideration the fact that the present study was conducted at high humidity (39e79%) and temperature (22e38 C), the findings may be readily applicable in countries with similar climatic characteristics, but not in colder and drier regions of the world such as northern Europe. Hydrophobic interactions play an important role in the nonspecific adherence of micro-organisms to biotic and abiotic surfaces.11 Thus, high bacterial hydrophobicity may favour the colonization, permanence and dissemination of pathogens in hospital environments.10,11 HI may vary between strains; therefore, five isolates of each bacteria (MSSA, MRSA and VISA) were also included in the hydrophobicity tests. All three test strains and the additional isolates tested were highly hydrophobic (HI >50%) (Table I). The results for VISA are particularly interesting as, to the authors’ knowledge, no data have been reported previously.
350
M.N. Zarpellon et al. / Journal of Hospital Infection 90 (2015) 347e350
The high hydrophobicity of VISA seems to be associated with its high surface adherence to vinyl flooring and formica, and the survival pattern observed. Under hot and humid conditions, cotton fabric would tend to absorb moisture, and this may explain why VISA (HI >89.57%) did not survive for long on this material. On the other hand, MSSA and MRSA also showed high HI but with different patterns of adherence to abiotic surfaces. In this study, the results from the hydrophobicity tests could not be related to the other parameters assessed (survival, adherence to abiotic surfaces and biofilm production). No adherence was observed on granite and little adherence was observed on glass, indicating that these types of materials could be considered for furniture or other surfaces in hospital settings. Due to the shortage of studies on the adherence of bacteria such as VISA to the materials used in hospital surfaces, further research is necessary to fully understand why some surfaces are much less favourable to VISA than others. Surface adherence is the preliminary factor for some microorganisms to form the complex extracellular matrix of polymeric substances that constitute biofilms.4,10,11 Biofilmforming bacteria may resist death by antibiotics, biocides and disinfectants compared with planktonic cells.5,10,12 The present study found that MRSA produced more biofilm compared with the other two test strains (Figure 1). These data were obtained on a polystyrene surface at 37 C with aeration, and may not be generalizable to other conditions, as the amount of biofilm formed by the different strains may vary between surfaces and environmental conditions.5,10 To the best of the authors’ knowledge, this is the first study to demonstrate that VISA presents high survival, adherence and cell-surface hydrophobicity. Therefore, as the treatment of patients with VISA is a significant challenge for clinicians, greater care with cleaning and disinfection of different types of surfaces in healthcare facilities is recommended as these may become important reservoirs of multi-resistant pathogens.
Acknowledgements The authors wish to thank Antonio Carlos Correa and Michael Arends for English text review. VISA were kindly provided by Ana Cristina Gales and Alessandro Conrado de Oliveira Silveira. Conflict of interest statement None declared.
Funding source This study was supported by the Coordenac ¸a ˜o de Aperfei¸ coamento de Pessoal de Nı´vel Superior and Fundac ¸a ˜o Arauca ´ria.
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