© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12254

APMIS 122: 1013–1019

Antiadhesive and antibiofilm activity of hyaluronic acid against bacteria responsible for respiratory tract infections LORENZO DRAGO,1,2 LAURA CAPPELLETTI,1 ELENA DE VECCHI,1 LORENZO PIGNATARO,3 SARA TORRETTA3 and ROBERTO MATTINA4 1 Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan; Laboratory of Technical Medical Sciences, Department of Biomedical Science for Health, University of Milan, Milan; 3Department of Specialistic Surgical Sciences, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan; and 4Department of Public Health, Microbiology and Virology, University of Milan, Milan, Italy

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Drago L, Cappelletti L, De Vecchi E , Pignataro L, Torretta S, Mattina R. Antiadhesive and antibiofilm activity of hyaluronic acid against bacteria responsible for respiratory tract infections. APMIS 2014; 122: 1013–1019. To address the problem of limited efficacy of existing antibiotics in the treatment of bacterial biofilm, it is necessary to find alternative remedies. One candidate could be hyaluronic acid; this study therefore aimed to evaluate the in vitro antiadhesive and antibiofilm activity of hyaluronic acid toward bacterial species commonly isolated from respiratory infections. Interference exerted on bacterial adhesion was evaluated by using Hep-2 cells, while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet. Our data suggest that hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentrationdependent manner, being notably active when assessed as pure substance. Moreover, we found that Staphylococcus aureus biofilm was more sensitive to the action of hyaluronic acid than biofilm produced by Haemophilus influenzae and Moraxella catarrhalis. In conclusion, hyaluronic acid is characterized by notable antiadhesive properties, while it shows a moderate activity against bacterial biofilm. As bacterial adhesion to oral cells is the first step for colonization, these results further sustain the role of hyaluronic acid in prevention of respiratory infections. Key words: Hyaluronic acid; bacterial biofilm; respiratory tract infections. Lorenzo Drago, Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Institute, Via R. Galeazzi 4, Milan, Italy. e-mail: [email protected]

According to the National Institutes of Health, up to 80% of human bacterial infections involve biofilm-associated microorganisms (1), and the hypothesis that bacterial biofilm might be implicated in the pathogenesis of respiratory tract infections (RTIs) has been supported by several studies (2–5). A biofilm is a complex organization of microorganisms attached to a surface and embedded in a slime-like matrix composed of polysaccharides, nucleic acids, and proteins known as extracellular polymeric substance (EPS) (6). Biofilm formation makes the bacteria resistant to host defense mechanisms as bacteria aggregated into Received 20 August 2013. Accepted 2 January 2014

EPS-coated biofilms become too large to be phagocytized (7) and are partly protected from the humoral immune system. In addition, the matrix protects the biofilm cells from various microbicidal agents and stresses, including dehydration, toxins, ultraviolet light, chemical disinfectants, temperature and osmotic shock (3), and lead them to increased resistance against antimicrobials (6). To address the problems of resistance to antimicrobials and the limited efficacy of existing antibiotics in the treatment of bacterial biofilms, novel approaches are required to prevent or treat infections. Adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new therapies (8). 1013

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Some specific biological mechanisms are used by bacteria for attachment to human cells, including receptors that are able to bind to specific ligands on the cell surface (8). Thus, bacterial colonization can be blocked by an inhibitor interfering with ligand–receptor interaction for bacterial attachment (9, 10). One of these inhibitors could be Hyaluronic Acid (HA), a glycosaminoglycan made up of glucuronic acid and N-acetylglucosamine disaccharide units. It is a uniform, linear and unbranched molecule, with highly variable length and molecular weight (up to 106 Da). It is abundant in skin (up to 56%) and in connective tissues, with a turnover ranging from several hours to a few days depending on tissues. Moreover, HA constitutes one of the main components of extracellular matrices (11). Because of its biological properties, HA has several clinical applications (aesthetic surgery, dermatology, orthopedics and opthalmology). In spite of such a wide employment, only few studies have been performed to assess the effects of HA against infectious agents (11). Some of them have provided evidence that HA is able to exert bacteriostatic effects against pathogens found in the oral cavity, depending on HA concentration and molecular weight (12, 13), but, to our knowledge, HA interference on bacterial adhesion and biofilm formation has not yet been thoroughly studied. Therefore, the aim of this study was to evaluate the antiadhesive and antibiofilm in vitro activity of HA toward bacterial species commonly isolated from patients with RTIs. MATERIALS AND METHODS Bacterial strains Five isolates each of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus isolated from patients with RTIs (age: 6–52 years; 12 males and 8 females) were included in the study. The isolates presented different antimicrobial patterns and were clinically isolated at different times.

Ability to produce biofilm The ability of each bacterial strain to produce biofilm was evaluated by a spectrophotometric method according to Christensen et al., (14). Briefly, biofilm formation was achieved by dispensing 20 lL aliquots of overnight broth cultures into wells of a 96-well polystyrene microtitre plate containing 180 lL of Tryptic Soy Broth (TSB) for S. aureus, TSB with 5% blood for M. catarrhalis and S. pneumoniae, and Haemophilus Test Medium (HTM) for H. influenzae. The test was performed in triplicate for each bacterial strain. After incubation for 24 h at 37 °C in presence (M. catarrhalis, H. influenzae and S. pneumoniae) or absence (S. aureus) of 10% CO2, non-adherent bacteria were removed. Then,

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new growth media were added and plates were further incubated for 48 h to obtain biofilm. Negative controls wells containing 200 lL of uninoculated broth were also prepared and incubated under the same conditions. At the end of the incubation time, medium was removed and three washes with Phosphate Buffered Saline (PBS) were performed to remove bacteria not included in biofilm. After air drying, each well was stained with 200 lL of 1% crystal violet solution for 10 min, then the excess dye was removed with three washes with PBS. Once dried, 200 lL of absolute ethanol was added to each well to solubilize the dye attached to the biofilm. The amount of biofilm produced was determined by spectrophotometric reading against blank (consisting of ethanol) at a wavelength of 590 nm, using a microplate reader (Multiskan Fc model; Thermo Scientific, Rodano, Italy). Comparing the optical density (OD) produced by bacterial biofilm to the OD of the negative control, it was possible to classify bacterial strains as non-producers (if OD590 of biofilm was lower than or equal to OD590 of the negative control), weak (if OD590 of biofilm ranged from OD590 of the negative control to twice this value), moderate (if OD590 of biofilm ranged from two to four times the OD590 of the negative control), or strong (if OD590 of biofilm was greater than four times the OD590 of the negative control) producers of biofilm (14).

Chemical Compounds Hyaluronic acid 0.3% (YABROâ; IBSA Farmaceutici Italia srl, Lodi, Italy) was tested at a concentration of 100% and 50% (v/v).

Interference on bacterial growth Before carrying out tests for evaluation of antiadhesive and antibiofilm activity of HA, studies were conducted to assess the effect of HA on bacterial growth. Bacteria (105 CFU/mL) were inoculated in 10 mL of 50% HA or in 10 mL of appropriate medium [Brain Heart Infusion broth (BHI), BHI with 5% blood or HTM] as positive control and incubated at 37 °C in the presence (M. catarrhalis, H. influenzae and S. pneumoniae) or absence (S. aureus) of 10% CO2. One hundred microliters of each bacterial culture was plated on blood agar or chocolate agar plates to verify the starting number of bacteria per mL. At different times (0, 30 min, 1, 2, 3, 4, 5, 6, 7, 8, 16 and 24 h), aliquots of 100 lL from broth cultures were transferred to a 96-microtiter plate and the absorbance at 600 nm was determined using a microplate reader (Multiskan Fc model; Thermo Scientific).

Interference on bacterial adhesion Interference exerted by HA on bacterial adhesion was evaluated by using an in vitro experimental model involving Hep-2 cells. Briefly, Hep-2 cells (Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia, Brescia, Italy) were seeded on CultureSlides (BD Falcon, Bedford, MA, USA) containing Dulbecco Modified Eagle medium (DMEM Low Medium, w/L-Glut; Sigma-Aldrich, Milan, Italy), 10% fetal calf serum (FCS; Sigma-Aldrich) and 1% of © 2014 APMIS. Published by John Wiley & Sons Ltd

HYALURONIC ACID IN RESPIRATORY INFECTIONS

penicillin/streptomycin (Sigma-Aldrich), and incubated at 37 °C in a humidified atmosphere of 5% CO2 in air until confluence. Antiadhesive activity of HA against the above-mentioned microorganisms was evaluated by co-incubating cells and bacteria previously treated with HA at the chosen concentrations. In particular, bacteria were resuspended in HA solution (100% or 50%) or PBS (negative control) to obtain 0.5 McFarland turbidity (1– 1.5 9 108 CFU mL 1) and 1 mL of such bacterial suspension was immediately incubated with Hep-2 cells grown at confluency. After 1 h of incubation grown at 37 °C in a humidified atmosphere of 5% CO2 in air, non-adherent bacteria were eliminated by three washings. Slides were then dried and Gram stained. The mean number of bacteria adherent per cell was determined by counting the number of bacteria adhering to 40 cells. Each test was performed in triplicate. Inhibition of adherence (IA) was calculated using the following formula: IA (%) = (100 mean number of treated bacteria adhered per cell/mean number of untreated bacteria per cell) 9 100.

Evaluation of activity against pre-formed biofilm Biofilm was allowed to form for 72 h prior to the addition of HA at the final concentration of 100% or 50% (v/v) as described above. Following the 48-h incubation period, HA was added to bacterial biofilm. The plates were further incubated for 3, 6 and 18 h before the spectrophotometric assay of biofilm (as described above). Untreated biofilm incubated in the same conditions was used as control. The mean absorbance of three replicates was determined and the inhibition rate was calculated by using the following formula: [(OD590 Growth control OD590 Sample)/OD590 Growth control] 9 100. Results were expressed as reduction (%) of biofilm in respect to positive controls.

Statistical analysis The mean of the three measurements for each experimental condition and control was taken as the outcome value.

RESULTS Ability to produce biofilm

Table 1 reports the mean absorbance values measuring biofilm produced by each bacterial strain. All S. aureus strains were strong producers of biofilm, while there were both strong and moderate producers among the H. influenzae and M. catarrhalis strains. S. pneumoniae strains were non-producers or weak producers; therefore, the antibiofilm activity of HA was not assessed with any of the pneumococcal isolates. Interference on bacterial growth

Figure 1 reports the growth curve of bacterial strains in the absence or presence of 50% HA in © 2014 APMIS. Published by John Wiley & Sons Ltd

Table 1. OD590 of Bacterial biofilm Bacterial strain S. aureus 1 S. aureus 2 S. aureus 3 S. aureus 4 S. aureus 5 Negative control H. influenzae 1 H. influenzae 2 H. influenzae 3 H. influenzae 4 H. influenzae 5 Negative control M. catarrhalis 1 M. catarrhalis 2 M. catarrhalis 3 M. catarrhalis 4 M. catarrhalis 5 Negative control + 5% blood) S. pneumoniae 1 S. pneumoniae 2 S. pneumoniae 3 S. pneumoniae 4 S. pneumoniae 5 Negative control + 5% blood)

(BHI)

(HTM)

(BHI

(BHI

Biofilm absorbance (mean  SD) 1.32  0.15 1.54  0.17 0.98  0.12 0.87  0.14 1.26  0.14 0.11  0.03 0.64  0.08 0.97  0.14 2.93  0.22 1.48  0.12 0.75  0.13 0.18  0.06 0.71  0.12 0.62  0.09 0.76  0.10 0.58  0.06 0.83  0.16 0.26  0.06 0.32 0.21 0.23 0.28 0.22 0.26

     

0.07 0.04 0.05 0.05 0.06 0.06

broth. No marked differences in growth curve were observed between treated and control bacteria. Interference on bacterial adhesion

Reduction in adhesion to Hep-2 cells of S. aureus, S. pneumoniae, M. catarrhalis and H. influenzae in respect to control when incubated with different concentrations of HA is reported in Fig. 2. When assessed as pure substance, HA presented a high antiadhesive activity against all the tested bacteria (adhesion reduction >70%), in particular against S. aureus (~ 90% of adhesion reduction compared to control), while at a concentration of 50%, the inhibition of bacterial adhesion ranged from 33% (H. influenzae) to 50% (S. aureus). Furthermore, microscopic analysis of treated cells did not show any sign of toxicity due to the action of HA: cells were living and well adherent to the glass surface. Interference on bacterial biofilm

Figure 3 shows the reduction of biofilm formed by S. aureus, M. catarrhalis and H. influenzae when incubated for 3, 6 and 18 h with different concentrations of HA compared with the control. The antibiofilm activity of HA was remarkable against S. aureus, especially after 18 h of incubation with 100% of HA (56% of biofilm reduction), while

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A

B

C

D

Inhibition of adherence (%) vs control

Fig. 1. Growth curve in presence or absence of 50% Hyaluronic acid (HA). (A) Staphylococcus aureus; (B) Haemophilus influenzae; (C) Moraxella catarrhalis; (D) Streptococcus pneumoniae. Continuous line: Control medium; Dashed line: HA 50%.

100 90 80 70 60 50 40 30 20 10 0

S. aureus

H. influenzae M. catarrhalis S. pneumoniae

Fig. 2. Interference of Hyaluronic acid (HA) on adhesion of Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae to Hep-2 cells. Black bars: hyaluronic acid 50%; White bars: HA 100%.

HA was found to be less active against biofilm formed by M. catarrhalis and H. influenzae, even after 18 h of incubation at a concentration of 100% (about 30% of biofilm reduction for both species).

DISCUSSION Due to the excessive use of antibiotics, in the last years, antibiotic resistance is spreading dramatically 1016

(15, 16). Therefore, it is necessary to find alternative remedies to prevent and treat RTIs. These alternative substances should be characterized by the ability to interfere with the adhesion of bacteria to host surfaces such as the epithelial cells of the respiratory tract, because this is the first important step in colonization and establishment of RTI (8). Another advisable requisite should be to exert some antibiofilm activity. In fact, it has been shown in several studies that biofilm plays an important role in etiology of numerous RTI (2, 3, 17, 18). Antibiotic therapy typically resolves symptoms determined by planktonic cells released by biofilms, but is often unable to eradicate and completely remove biofilm (19). This is one of the factors leading to recurrence of infections sustained by biofilm-producer bacteria, necessitating repeated antibiotic treatments and facilitating the development of antibiotic resistance (20). A potentially alternative as a coadjuvant remedy against the adhesion of bacteria in RTIs might be HA, a structural component of interstitial and connective tissues. Extensive studies on the chemical and physicochemical properties of HA and its physiological role in humans, together with its versatile properties, such as its biocompatibility, non-immunogenicity, biodegradability, and viscoelasticity, have proved that it is an ideal biomaterial © 2014 APMIS. Published by John Wiley & Sons Ltd

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A

B

C

Fig. 3. Reduction of bacterial biofilm by Hyaluronic acid (HA). (A) Staphylococcus aureus; (B) Haemophilus influenzae; (C) Moraxella catarrhalis. Continuous line: HA 100%; Dashed line: HA 50%.

for medical and pharmaceutical applications (21). It is used for supplementation of impaired synovial fluid in arthritic patients, following cataract surgery, as a filler in cosmetic and soft tissue surgery, as a device in several surgical procedures, particularly as an antiadhesive following abdominal procedures, and also in tissue engineering (22). Zahm et al. (23) demonstrated that HA plays a critical role in the airway epithelial integrity homeostasis and in the protection against the deleterious effect of virulence factors secreted by bacteria during infection. Therefore, they suggested that HA may play a possible therapeutic role in a variety of respiratory diseases. Then, the main purpose of this study was to evaluate the antiadhesive and antibiofilm in vitro activity of HA toward bacterial species commonly involved in RTIs, to verify this possible therapeutic © 2014 APMIS. Published by John Wiley & Sons Ltd

role. First of all, however, studies were conducted to assess the effect of HA on bacterial growth. Results obtained suggested that HA is unable to affect the lag phase of bacterial growth. These data are in contrast with those reported by other authors (12, 13), but this discrepancy might be due to the different experimental conditions used (different concentrations and formulation of HA, various bacterial inocula). Our data suggest that HA is able to interfere with bacterial adhesion to a cellular substrate in a concentration-dependent way, being notably active when assessed as pure substance. This ability was observed against all the four bacterial species tested, thus indicating a non-specificity of action of HA. As the growth curves showed that HA did not interfere with bacterial multiplication, we can suppose that the presence of a lower number of bacteria adherent to the Hep-2 cells was not due to a toxic effect of HA on bacterial cells. Therefore, further studies will be needed to understand how HA prevents bacterial adhesion to a cellular substrate. Hyaluronic Acid has been shown to exert varied bacteriostatic but not bactericidal effects on oral and non-oral microorganisms in the planktonic phase (12), but there are no data about the sessile phase (biofilm). As it is well known that bacteria embedded in biofilms are very resistant to antibiotics, capable of surviving to antibiotic concentrations thousands of times greater than planktonic bacteria (6), development of antimicrobial compounds that are able to disaggregate biofilm matrix, thus allowing to antibiotics to reach their target at optimal concentrations, may represent a further aid in treatment of biofilm-associated infections. We found that S. aureus-biofilm was quite sensitive to the action of HA, while biofilm produced by the Gram-negative bacteria tested in this study seemed to be less susceptible to the activity of HA, although a reduction of up to 30% was observed after 18 h of incubation. It would be interesting to understand the reason behind this different susceptibility. The biofilm matrix is defined as the polymeric material that holds the community of bacterial cells together on a surface. Depending on bacterial species, strain type and environmental conditions, the biofilm matrix consists of substances of various chemical nature, such as exopolysaccharides, proteins, teichoic acids and extracellular DNA (eDNA) (24). Therefore, we can suppose that the matrix-composition of biofilm produced by strains of S. aureus is different from that of other species tested and this could explain the conflicting results of treatment with HA. From this point of view, further studies to better clarify how HA acts on biofilm are advisable.

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A limitation of this study is that the strains used were not tested for the ability to produce hyaluronidase, an enzyme that catalyzes the degradation of HA: in fact, it is reported that most of S. aureus and S. pneumoniae strains are able to produce such enzyme (25). Therefore, it is possible that in some cases, results obtained on antibiofilm and antiadhesive activity of HA were underestimated due to the cleavage of HA by hyaluronidase. Another limitation could be the use of Hep-2 cells to evaluate interference of HA on bacterial adhesion. This cell line was derived from the larynx, but it has been reported that it was probably contaminated with HeLa cells (26). However, use of Hep-2 cells for the study of bacterial-adhesion for the respiratory cells has been widely reported (27, 28). In conclusion, HA is characterized by notable antiadhesive properties, while it shows a moderate activity against bacterial biofilm (except for S. aureus). As bacterial adhesion to oral cells is the first step for colonization, these results further sustain the role of HA in prevention of RTIs.

We thank dr Christian Vassena for laboratory assistance.

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25. Hynes WL, Walton SL. Hyaluronidases of gram-positive bacteria. FEMS Microbiol Lett 2000;183:201–7. 26. Kennedy GE. Origin of Hep-2 cells used for culture of chlamydiae. J Clin Microbiol 1993;31:470–1. 27. Murakami J, Terao Y, Morisaki I, Hamada S, Kawabata S. Group A streptococcus adheres to pharyngeal epithelial cells with salivary proline-rich

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Antiadhesive and antibiofilm activity of hyaluronic acid against bacteria responsible for respiratory tract infections.

To address the problem of limited efficacy of existing antibiotics in the treatment of bacterial biofilm, it is necessary to find alternative remedies...
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