Identification of Factors Associated with Biofilm Formation Ability in the Clinical Isolates of Helicobacter pylori

Background A few reports confirm the ability of Helicobacter pylori to form biofilm. However, conclusive data do not exist concerning the factors that favor this ability. Objectives Evaluation of the factors associated with the biofilm formation ability of H. pylori including bacterial, physical and chemical, and environmental factors was the research’s aim. Materials and Methods H. pylori isolates from gastric biopsy specimens of patients infected chronically were screened for biofilm formation ability. Association of bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and extracellular polymeric substances (EPS) with in vitro biofilm formation ability of H. pylori was evaluated. The effects of environmental factors such as growth-medium, temperature, oxygen-tension, pH, β-cyclodextrin, gastric secreted mucins, and sub-inhibitory concentration of amoxicillin were also evaluated. Results Ability of clinical H. pylori isolates to form biofilm in was quantitatively compared. The coccoid shape H. pylori cells were observed by scanning electron microscopy, the images were illustrative of the attachment of cells to form microcolony. The levels of hydrophobicity, motility and auto aggregation of two isolates with highest and lowest biofilm formation ability were the same. However, the signifi cant role of mucins (P < 0.05) in elevating the biofilm formation was observed. Other factors influencing biofilm formation were: pH, atmosphere and sub-MIC of antibiotics. Conclusion Mucins have a signifi cant role in elevating the biofilm formation, also pH, atmosphere and sub-MIC of antibiotics influence biofilm formation.


Background
Infection by Helicobacter pylori is associated with gastritis and peptic ulcer disease and may be a risk factor for gastric carcinoma and MALT lymphoma (Mucosa-associated lymphoid tissue) (1,2).
The biofi lm mode of growth is a survival strategy deployed by many bacteria and is manifested as communities of cells attached to each other and/or to surfaces or interfaces, which are embedded in a selfproduced matrix of extracellular polymeric substances (EPS) (3)(4)(5). Although biofi lm formation would be slower in vivo than in vitro, once formed it may be able to induce the signaling process that conduct to the transcriptions of the bacterial genes, which are not expressed in planktonic cells. These signaling events may also be dependent on the nature of bacterial microenvironments, which can prevent or enhance its interaction with the host (6,7).
For a gastric pathogen such as H. pylori the host microenvironment would be very diff erent from that of the exterior. After entry, H. pylori is surrounded by the host microenvironment, which contains mucins as integral part of the stomach mucosal barrier. Hence, the microenvironment surrounding the bacteria may also play a role in favoring or preventing production of the biofi lm (8). The fi rst report from the ability of H. pylori to form a biofi lm indicated that this behavior may facilitate survival of bacteria in the stomach (9). Later studies indicated that bacterial biofi lms are embedded in a self-produced extracellular matrix, which is a complex mixture of exopolysaccharides, proteins, DNA and other macromolecules (10). Furthermore, a

Objectives
For a pathogen such as H. pylori the bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and presence of the exopolymeric matrix of biofi lms may be important in its survival and proliferation. Moreover, eff ects of some physical and chemical environmental factors such as temperature, pH, and aerobic or micoaerophil atmosphere or low concentrations of the antimicrobial agents are amongst the factors that H. pylori may encounter in its life cycle. For this purpose, these factors were evaluated by using of H. pylori isolates from chronic infection of children and adults, consisting of an effi cient biofi lm forming isolate and a weak biofi lm forming isolate.
Identifi cation of the eff ective factors involved in the biofi lm formation by H. pylori, may help to better prevent its formation in host stomach. Furthermore, determination of the biofi lm formation conditions, may help to select a better eradication regiments to circumvent biofi lm formation and so chronic infection by antibiotic resistant bacteria.

Bacterial Isolates and Growth Conditions
A collection of 25 clinical H. pylori isolates from the chronic infection of children and adults were plated onto modifi ed Campy blood agar containing brucella agar base (Merck, Germany), supplemented with 5% defi brinated sheep blood, and antibiotics (polymyxin B, amphotericin B, vancomycin), and incubated at 37°C under microaerobic atmosphere (10% CO 2 , 5% O 2 , and 85% N 2 ) for three days. The grown colonies were identifi ed by Gram staining, biochemical tests (catalase, oxidase, urease, nitrate) and PCR, using H. pylori-specifi c primers 16sRNA and ureC, as previously described (18).

Analysis of Biofi lm by Staining
The grown colonies were harvested from culture plates and inoculated into brucella broth (Biolife, Italy) supplemented with 2% (w/v) fetal calf serum and 0.3% (w/v) glucose (Merck, Germany). Bacterial suspensions were incubated at 37°C in a microaerobic atmosphere under shaking at 100 rpm for 16 h. Broth cultures were adjusted to an optical density of 0.2 at 600 nm (A 600 ) equivalent to a turbidity of 5-8 × 10 3 colony-forming unit (CFU).mL -1 , which corresponded to the beginning of exponential phase. Culture broth (250 μl) was inoculated into the polystyrene 96-well fl at-bottomed tissue culture plates (BIOFIL, Jet Bio-Filtration Products Co., Ltd). The plates were incubated at 37°C under microaerobic condition for 6 days (19). The plates were vigorously washed (thrice) with sterile phosphate-buff ered saline (PBS) (pH 7.2 -7.4) in order to remove all non-adherent bacteria, were fi xed with 99% ethanol for 20 min, air dried and stained with 1% Crystal Violet for 5 min; the excess of stain was rinsed away by running tap water and the dried plates were treated with 33% (v/v) glacial acetic acid, for solubilizing the attached dye. Optical density (OD) of the wells was measured at 505 nm according to the previously adopted protocol (20), using an ELISA reader (SCO, Germany).

Scanning Electron Microscopy of Biofi lms
Cell suspensions were inoculated into 12-well cell culture plates. A coverslip was inserted into each well and the plate was incubated at 37°C under microaerobic conditions for 3 and 6 days. Thereafter, the coverslips were removed from the wells and rinsed off (by dipping in sterile medium) to remove non-adherent cells. They were fi xed in 2% glutaraldehyde for 2 h and washed in PBS. The samples were dehydrated in a graded ethanol series of 25, 50, 75, 95, and 100% and stored in the desiccators until they were coated with gold-palladium sputter for two 200-second intervals (Nano Structured coating Co. Iran). SEM micrographs were performed using TESCAN VEGA3S electron microscope, at 30 KV.

Cell Viability Assay
Bacterial culture (2 mL) were seeded in 12-well culture plates (Nunc, Denmark), according to the protocol noted above, washed (thrice), and incubated with brucella broth, supplemented with 0.05% (w/v) of 2, 3, 5 triphenyl tetrazolium chloride (TTC) (Merck, Germany), at 37°C under microaerobic conditions. After 24 h, the broth was removed, the wells were air dried and the bound TTC dye was dissolved using 20% acetone -/80% ethanol; OD was then measured at 505 nm (21). To determine the number of biofi lm forming bacteria, seeded culture plates were washed with sterile PBS (500 μL), the biofi lm was removed with an ultrasonic bath for 7 min (Elmasonic S 60/ (H) - Germany, Ultrasonic frequency: 37 kHz), and CFU was determined. The results were presented as the mean of three independent tests (22).

Motility Assay
Motility of the H. pylori isolates were assessed by the method of Tan et al. (23). Bacteria were incubated in brucella broth supplemented with 3% serum was collected after 24 h, resuspended in PBS and the optical density (OD 600 ) was adjusted to 1.0. Motility agar plates (0.35% agar medium containing brucella broth supplemented with serum), were inoculated with 5 μL of the bacterial suspension, and incubated under microaerobic condition at 37°C. The diameter of halos produced after 3-5 days were measured using three plates for each case.

Analysis of Biofi lm Formation at the Air-Liquid Interface
A coverslip was placed into the wells of 12-well culture plates previously seeded with the bacterial cells as noted above, covered and incubated at 37°C under microaerobic conditions. After 3 and 6 days incubation, the coverslips were removed, rinsed off in sterile medium and the cells were stained by submersion in 0.1% crystal violet for 10 min. The biofi lms were visualized by inverted microscope (24).

Cell Surface Hydrophobicity
Cell surface hydrophobicity was performed according to the method of Yonezawa and Osaki (11). Overnight bacterial culture (1 mL) in brucella broth was added to 9 mL of fresh medium and was incubated at 37°C for 20 h. Bacteria was harvested by centrifugation, washed (3) with PUM buff er (0.15 mol.L -1 potassium phosphate buff er, pH 7.1, containing 0.3 mol.L -1 urea and 6.7 mmol.L -1 MgSO 4 ), and resuspended in the same buff er. The resulting suspension was adjusted to OD 400 1.0, to which 600 mL of n-hexadecane (Merck, Germany) was added, and mixed vigorously for 60 s. After 15 min incubation at 22°C, the absorbance of the aqueous layer was measured at 400 nm. The cell surface hydrophobicity was expressed as follows: (OD 400 before mixing -OD 400 after mixing) /OD 400 before mixing × 100.

Auto-Aggregation Assay
Auto-aggregation assay was determined using a method adopted from Yonezawa et al. (11). Bacterial culture was washed, resuspended in PBS, adjusted to OD 600 1.0 and incubated at 22°C. ODs were read over time at 600 nm. The percent of auto-aggregation was measured as follows: Auto-aggregation = (pre-incubation value [OD 600 ] -incubation value [OD 600 ]) / (pre-incubation value [OD 600 ] × 100.

Analysis of Extracellular Polymeric Substances (EPS)
Bacterial biofi lms produced in 12-well cell culture plates (as noted above), were washed (thrice) with sterile distilled PBS and the cells were removed by incubation in an ultrasonic bath (Elmasonic S 60/ (H)-Germany, Ultrasonic frequency: 37 kHz) for 7 min. The cell suspension was extracted with 2% EDTA for 4 h at 4°C, centrifuged at 10000 ×g, 4°C for 20 min and the supernatants were fi ltered through a 0.22 μm polyvinylidene membrane (Fluoride, Millipore). The fi ltrates were used for determination of polysaccharide and protein contents according to the method adopted by Pan et al. (25). Polysaccharide content of EPS was determined by the phenolsulphuric acid method, according to Dubois and Gilles (26); glucose was used as the standard. Protein content of EPS was determined by the Bradford method (27) and the bovine serum albumin was used as the standard. In this analysis, the EPS of S7 clinical isolate of Pseudomonas aeruginosa was used as control (28).

10. Eff ect of Mucins on Biofi lm Formation
To evaluate the eff ect of native mucins (porcine mucins, Sigma Aldrich, USA) on biofi lm formation, it was tested under various experimental conditions. First, various concentrations of mucins sterilized by gamma irradiation (dose of 3.5 kGy), was added to the culture medium using the previously described method (29), and the results of biofi lm formation was compared. In the second case, the culture plates were coated with 1 mg/well of mucins (30), washed three times with PBS-0.1% Tween-20, after overnight incubation at 37°C; the plates were then stored at 4-8°C until use. Quantitative detection of biofi lm was performed by staining of the plates by crystal violet, Safranin also by TTC, followed by measurement of OD by ELISA reader.

11. Evaluation of the Various Factors Aff ecting Biofi lm Formation Using the Placket-Burman Design
Multiple factors that may aff ect the behavior of H. pylori, including growth-medium, addition of glucose, temperature, oxygen-tension, pH, -cyclodextrin, mucins, and sub-inhibitory concentration of amoxicillin on the biofi lm formation by H. pylori were studied according to placket-Burman design using Minitan16 software as demonstrated in Table 1.
The non-favorable temperature (28°C) was compared with 37°C; as well as the non-favorable atmosphere (aerophilic) was compared with microaerobic atmosphere; the very acidic (pH 2) was compared with pH 7. The eff ects of various media (brucella broth and F12) (31,32) in biofi lm formation as well as the addition of glucose (2%) were evaluated (9). -cylodextrin (12) and sub inhibitory concentration of amoxicillin were selected as the examples of the factors that may produce the favorable or non-favorable conditions for H. pylori. Each additional factor was added to the medium after sterilization (0.45 μm polyvinylidene fl uoride, Millipore,). The role of mucins presence was also included in this design.

1Statistical Analysis
Statistical analysis was carried out using ANOVA one-way test with Minitab 16 statistical software, and probability levels of <0.05 were considered as statistically signifi cant. For in vitro studies, three independent experiments with eight replicates were performed and in each test, the broth without bacteria was used as the negative control.

In vitro Biofi lm Formation by H. pylori Clinical Isolates
Screening of the clinical isolates for biofi lm forming ability showed that under favorable experimental conditions (brucella broth supplemented with 2% (w/v) fetal calf serum and 0.3% (w/v) glucose and incubation at 37°C in a microaerobic atmosphere with shaking at 100 rpm for 6 days) almost all of the isolates formed relatively little biomass on plates. Amongst which two isolates with signifi cantly higher (19B) and lower (4B) levels of biofi lm ability were selected (Fig. 1). 19B was isolated from an 11years-old female patient with endoscopic status of severe gastritis and pathology of moderate active chronic gastritis. 4B was isolated from a 38 years-old male patient with endoscopic status of normal and pathology of mild chronic gastritis.

Scanning Electron Microscopy of Biofi lm
SEM micrograph of biofi lm forming cells after 6 days (Fig. 2) demonstrated that a tight attachment of  coccoid bacterial cells was formed in microcolonies. This morphology could be compared with that of the planktonic populations recovered from the supernatant of the same isolate.

Cell Viability Assay
Viability of the biofi lm forming cells was evaluated after 3-day and 6-day by two methods of viable counting (CFU) and straining method using Crystal Violet as well as TTC. Comparison of the staining results for two periods demonstrated that OD of TTC-stained cells were almost similar (Table 2). Similarly, the number of bacteria counted by CFU method was also comparable for two periods.

Air-Liquid Interface Coverslip (CV) Assay
Both isolates formed a biofi lm at air-liquid interface on coverslip surface. However, 19B formed denser community on the glass surface compared to 4B.

Motility, Hydrophobicity, and Auto-Aggregation Assays
19B and 4B were both motile with no signifi cant diff erences between their motility halos. Their hydrophobicity levels were also similar (63.7 ± 0.452 for 19B and 68.36 ± 1.108 for 4B). Concerning their auto-aggregation abilities, both isolates showed strong auto-aggregation ability with no signifi cant diff erence (Fig. 3).

Evaluation of Polysaccharide and Protein Contents in EPS
By comparison of protein and polysaccharide content in the EPS of H. pylori isolates, 19B and 4B, expressed in their percentage (relative to the total weight of the biofi lm), no signifi cant diff erences were observed between the two. However, their percentages were signifi cantly higher in Pseudomonas aeruginosa S7, in comparison with 19B and 4B.

Eff ect of Mucins on Biofi lm Formation
Addition of various concentrations of mucins to the culture medium, had no signifi cant eff ect on the biofi lm formation ability; but coating of plates with mucins before bacterial loading, demonstrated a signifi cant positive eff ect on the biofi lm formation by H. pylori (Fig. 4).

Placket-Burman Design
The eff ects of growth medium, addition of glucose to the growth medium, temperature, pH, sub-MIC concentration of amoxicillin, -cyclodextrin, various incubation atmosphere (oxygen tension), and presence of mucins, on H. Pylori biofi lm formation is demonstrated in Figure 5. Among various favorable factors atmosphere (microaerobic), pH 7.0 , presence of mucins and sub-MIC concentration of amoxicillin had signifi cant eff ects (p < 0.005) on biofi lm formation by H. pylori.

Discussion
Signifi cance of eff ective factors in biofi lm formation of H. pylori has not been well evaluated. By selecting a pair of strains consisting of an effi cient biofi lm-forming and a weak biofi lm-forming, important factors were evaluated. Through understanding the factors that aff ecting biofi lm formation by this pathogen, one may encounter the H. pylori and stop the biofi lm formation in the future. Initial evaluation of biofi lm formation by H. Pylori clinical isolates were performed by crystal violet staining method. For quantitative evaluation of biofi lm formation and count of viable cells, an optimized TTC staining method was used (21). Its advantage   is that metabolically active bacteria reduce 2, 3, 5 triphenyltetrazolium chloride (TTC) to insoluble, red crystals of 1, 3, 5-triphenylformazan (TFP), which can be measured calorimetrically. TTC data were more consistent with those of CFU (Table 2). It seems that TTC staining is an accurate method to evaluate both biofi lm formation by H. pylori and its viability.
Assessment of biofi lm formation by electron microscopy in 19B and its planktonic populations showed tight adherence between biofi lm forming cells, which lead to the microcolony formation (Fig.  2). Presence of coccoid form cells, which were fi rmly attached together, suggests that this state may be dominant in the H. pylori biofi lm. This observation is consistent with other in vitro studies demonstrating that coccoid form H. pylori is present more in its biofi lm state (7). Consistent with an earlier report (12), H. pylori isolates used in this work were able to form a biofi lm at air-liquid interface on coverslip surface. However, the biofi lm of 19B demonstrated the dense communities attached to the glass surface.
Contrary to the results of Yonezawa et al. (11), no signifi cant diff erences were observed between the hydrophobicity levels, motility and auto-aggregation of 19B and 4B. This diff erence in the results may be related to the diff erences among the isolates employed in the two studies.
Potentially, the nature of EPS and its amount may play a role in biofi lm formation (25). Although resistance of biofi lm forming cells to antibiotics may be a very complex phenomenon, biofilm forming cells may be protected by their EPS via prevention of antimicrobial penetration (34).
In the case of H. pylori, only one report has evaluated its EPS, and demonstrated 1, 4-mannosyl linkages in H. pylori biofi lms and mannose as main sugar (35). We assessed the amount of polysaccharide and protein in EPS of H. pylori isolates and compared their contents with those of Pseudomonas aeruginosa S7 isolates (Table 3). Although their contents were lower than those of P. aeruginosa biofi lms, their signifi cant amounts indicated that such as the case of P. aeroginosa, the polysaccharide and protein content of EPS may be important for H. pylori.
The only one study analyzing the role of mucins in biofi lm formation by H. pylori reports no eff ect on the biofi lm formation or on the number of adherent bacteria (12). Contrary to this fi nding, our result showed that mucins signifi cantly increase biofi lm formation by H. pylori (Figs. 4 and 5). The eff ect of mucins on the adherence of bacteria to culture plates as well as on the number of biofi lm forming bacteria were analyzed. On the contrary to their method of sterilization which was by autoclaving that cause structure damage, we conserved the native structure of mucins by gamma irradiation using a dose of 3.5 kGy, which was proven to maintain its integrity (29).
To determine eff ective factors in biofi lm formation by this pathogen, Placket-Burman design was used. Accordingly, the positive role of four factors were demonstrated. The factors were microaerobic atmosphere, pH (pH 7), mucins and sub-MIC concentration of amoxicillin, as the favoring factors in the biofi lm formation (Fig. 5).
Presence of mucins or microaerobic conditions as well as sub MIC concentration of the antibiotic (due to inappropriate doses of antibiotics), may be amongst the factors that H. pylori may encounter in its life cycle. Also in natural conditions of stomach, H. pylori may prefer neutral condition to acidic pH for biofi lm formation.
Reuter and et al. (36) have shown that biofi lm formation by Campylobacter jejuni is enhanced under aerobic condition perhaps due to the fact that C. jejuni could adapt to the aerobic condition and survived in this atmosphere via biofi lm formation. Although this adaptation to the environment may be important for survival during transmission (36), we observed that H. pylori was not able to act similar to C. jejuni, perhaps due to its slower growth rate in aerobic conditions or its more sensitivity to oxygen pressure. A decrease in biofi lm formation at pH 2 was observed that suggests that the presence of an active urease is not solely suffi cient to circumvent the low pH. Since H. pylori uses its fl agella to reach the neutral environment of the gastric epithelial cells of distal stomach. Consistent with this suggestion, the non-motile mutants of H. pylori are less virulent. Furthermore, freshly obtained isolates from stomach had the spiral morphology, whereas those subcultured in the laboratory had bacilli or even coccoid morphology. Motility of the two isolates with high and low biofi lm forming ability were almost similar suggesting that for in vitro biofi lm formation, H. pylori does not require motility.
Using the sub MIC concentration of amoxicillin, an increase in biofi lm formation was observed. By creation of non-favorable conditions for bacteria, the sub-MIC concentration of amoxicillin favors biofi lm formation, which helps bacteria better resist this drug. Bessa et al. (32) have reported that sub-MICs of amoxicillin and clarithromycin increased biofi lm formation in H. pylori. This kind of resistance helps bacteria circumvent a stress condition or resist a drug, a situation that may be occurred in stomach since a biofi lm formed in vivo may be a real barrier to the antibiotics. Clarifi cation of this resistance in H. pylori may help to develop more eff ective treatment regimens as well as strategies other than the traditional means (37).
By screening of the clinical H. pylori, we demonstrated their ability to form a typical bacterial biofi lm in vitro. Also, by evaluating the role of various environmental factors, we could demonstrate the favorable factors that enhance the biofi lm formation ability in vitro. They include presence of mucins, favorable pH and microaerophil atmosphere and sub-MIC concentration of antibiotics, the roles which may be extrapolated to in-vivo conditions.
The results of present study that suggest favorable roles of multiple factors in biofi lm formation, may also be important in biofi lm formation by H. pylori in stomach. Evaluation of the factors that increase biofi lm formation by H. pylori in animal models may be useful in planning of the biofi lm formation prevention and as a result reducing generation of antibiotic resistance and chronic infection.