https://environhealthprevmed.biomedcentral.com/articles/10.1186/s12199-017-0670-3
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Enterococcus hirae biofilm formation on hospital material surfaces and effect of new biocides
Environmental Health and Preventive Medicine 22, Article number: 63 (2017)
Abstract
Background
Nowadays, the bacterial contamination in the hospital environment is of particular concern because the hospital-acquired infections (HAIs), also known as nosocomial infections, are responsible for significant morbidity and mortality. This work evaluated the capability of Enterococcus hirae to form biofilm on different surfaces and the action of two biocides on the produced biofilms.
Methods
The biofilm formation of E. hirae ATCC 10541 was studied on polystyrene and stainless steel surfaces through the biomass quantification and the cell viability at 20 and 37 °C. The effect of LH IDROXI FAST and LH ENZYCLEAN SPRAY biocides on biomasses was expressed as percentage of biofilm reduction. E. hirae at 20 and 37 °C produced more biofilm on the stainless steel in respect to the polystyrene surface. The amount of viable cells was greater at 20 °C than with 37 °C on the two analyzed surfaces. Biocides revealed a good anti-biofilm activity with the most effect for LH ENZYCLEAN SPRAY on polystyrene and stainless steel at 37 °C with a maximum biofilm reduction of 85.72 and 86.37%, respectively.
Results
E. hirae is a moderate biofilm producer depending on surface material and temperature, and the analyzed biocides express a remarkable antibiofilm action.
Conclusion
The capability of E. hirae to form biofilm can be associated with its increasing incidence in hospital-acquired infections, and the adoption of suitable disinfectants is strongly recommended.
Background
Nowadays, the bacterial contamination in the hospital environment is of particular concern because the hospital-acquired infections (HAIs), also known as nosocomial infections, are responsible for significant morbidity and mortality [1]. The European Centre for Disease Prevention and Control (ECDC) estimated that, in the EU, each year, about 4, 100,000 patients acquired a healthcare-associated infection, resulting in 110, 000 deaths [2].
According to ECDC, HAIs are infections contracted in the healthcare setting (e.g., inpatient hospital admission or same-day surgery) that can originate from different sources such as external environment, infected patients, healthcare staff that may be infected, contaminated items (food, water, medications, devices, and equipment), or droplets containing microbes [3, 4].
It is well known that bacteria, exposed to various stresses (e.g., antibiotics, nutrient limitation, non-permissive temperature), can express the ability to form multicellular organizations as a network of cell-to-cell interactions attached to each other and/or to surfaces, called biofilms that permit survival in adverse environments [5], and that are difficult to treat, resulting in an enormous impact on healthcare [6,7,8,9].
Therefore, the capability of bacteria to grow on biofilm mode surfaces is in another aspect to consider in the clinical contamination.
Actually, equipment for sanitation and hand-touch surfaces sanitizing/sterilization together with hand washing of visitors to patients, and all medical personnel are the most effective ways to contrast nosocomial infections [10]. Consequently, hospital disinfection policies play an mportant role in the control of HAIs [11, 12]. “Biocidal products are those that are intended to destroy, render harmless, prevent the action of, or otherwise exert a controlling effect on any harmful organism by chemical or biological means; examples include disinfectants, preservatives, antiseptics, pesticides, herbicides, fungicides and insecticides” (Biocides Directive 98/8/EC 1998) [13]. They are used widely for the disinfection of surfaces and equipment, and for sterilization of medical devices.
Enterococci, previously included among gut commensals of humans and animals, in the last years, have acquired the role of common nosocomial pathogens (the third leading source of nosocomial infection), causing urinary tract infections, endocarditis, peritonitis, and bacteremia [14]. Among these, Enterococcus hirae, proposed as a new test germ within the framework of the procedures for the European standardization of chemical and antiseptic agents for evaluation and validation of disinfectant products (EN14561:2006) [15], has been recently described as an emergent nosocomial pathogen in HAIs [16, 17]. Despite, human E. hirae infection is extended to be 1–3% of the Enterococcus spp. infections detected in clinical practice [18], and an emerging role of source of serious illness can be attributed to this microorganism responsible of endocarditis, acute pancreatitis, pyelonephritis, and septic shock [18,19,20,21,22,23]. In sight of this and taking into account the role that the biofilms have in hand-touch surface-associated nosocomial infection, our aim was to characterize the biofilm formation of E. hirae on polystyrene and stainless steel surfaces, and to investigate the antibiofilm action of two biocides, to be used in the disinfection of surgical and the medical device.
Methods
Strain culture condition
The reference strain E. hirae ATCC 10541 was used for this study. To recover the strain, a loop of cell was picked up from the strain stored at −80 °C and cultured in Tryptic Soy Broth (TSB, Oxoid, Milan, Italy) plus 1% v/v of glucose (Sigma Aldrich, Milan, Italy) (TSBG) at 37 °C overnight under aerobic condition. After incubation, the broth culture was diluted 1:10 in the same medium and refreshed for 2 h at 37 °C in shaking thermostat water bath (160 rpm). Finally, the culture was adjusted in spectrophotometer (Eppendorf, Milan, Italy) to optical density OD600 = 0.12 corresponding to 0.5 Mcfarland [24]. This broth culture standardized was used for the experiments.
In vitro biofilm formation and biomass quantification
The biofilm formation of E. hirae was evaluated on polystyrene and stainless steel, two surface materials widely used in the hospital, at two different incubation temperatures, 20 and 37 °C [25]. For the analysis on polystyrene surface, the standardized broth culture (200 μL) was inoculated on flat-bottomed 96-well polystyrene microtiter plates, and incubated at 20 and 37 °C for 48 h. After incubation, the planktonic cells were removed from each well and biofilms and the respective negative control (TSBG without bacteria) were rinsed with sterile water, fixed by air drying, and stained with Crystal Violet 0.1% (Sigma Aldrich, Milan, Italy) for 1 min. The stained biofilms were washed with sterile water and eluted with ethanol for reading.
For analysis on stainless steel surface, sample sheets (0.5 mm thickness) of stainless steel, obtained locally, were divided into small coupons (1 cm × 1.5 cm) and used as surfaces for biofilm growth. Prior to testing, stainless steel coupons were washed with detergent, rinsed with distilled water, immersed in 70% ethanol, rinsed again with distilled water, and finally sterilized [26].
Two microliters of E. hirae ATCC 10541 standardized broth culture were used to cover totally sterile stainless steel coupons, placed into Petri dishes (3.5 cm of diameter). Petri dishes were incubated aerobically at 20 and 37 °C for 48 h. After incubation, the planktonic bacteria were removed from Petri dishes and biofilms were washed with sterile water, dried as previously described [27] and stained with Crystal Violet 0.1% for 1 min, washed with sterile water, and eluted with ethanol. Two hundred microliters of eluted solution were read by using a microplate reader (SAFAS, Munich, Germany) with an absorbance of 595 nm.
Three independent experiments in triplicate, for each temperature and material surface, were performed.
Afterwards, using the OD595 measurements of biofilms formed, E. hirae ATCC 10541 was classified as strong, moderate, or weak biofilm producer according to Stepanovic et al. [28], as follows: OD ≤ O.D.c = no biofilm producer, O.D.c < OD ≤ (2× O.D.c) = weak biofilm producer, (2× O.D.c) < OD ≤ (4× O.D.c) = moderate biofilm producer and (4× O.D.c) ≤ OD = strong biofilm producer. The cut-off O.D.c was defined as three standard deviations above the mean OD of the negative control.
Action of biocides in the removal of biofilms formed on polystyrene and stainless steel
The two biocides were provided by Lombarda H S.r.l. (Albairate, Milan, Italy). The chemical characteristics are showed in Table 1, and they were used at concentration recommended by the biocide manufacture. The biofilms of E. hirae ATCC 10541 on polystyrene, and stainless steel were performed under the same conditions described above. After 48 h of formation at 20 and 37 °C, the planktonic cells were removed and the biofilms formed on materials were treated with biocides for 60 min. All experiments included controls (with biocides) and TSBG (without bacteria). After incubation, the treated biofilms were washed, stained with Crystal Violet 0.1% for 1 min, washed with sterile water, eluted with ethanol, and read as described above.
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