Changes in adhesion ability of Aeromonas hydrophila during long exposure to salt stress conditions

Authors


Correspondence

Anna Pianetti, Biomolecular Sciences Department, University “Carlo BO”, Via S. Chiara 27, 61029 Urbino, Italy. E-mail: anna.pianetti@uniurb.it

Abstract

Aims

Stressful environmental conditions influence both bacterial growth and expression of virulence factors. In the present study, we evaluated the influence of NaCl on Aeromonas hydrophila adhesiveness at two temperatures. This agent is often involved in clinical cases; however, its pathogenic potential is still not fully understood.

Methods and Results

Bacteria were grown in presence of 1·7%, 3·4%, 6·0% NaCl over a 188 day period and then reinoculated in fresh Nutrient Broth with incubation at 4 and 24°C. Bacterial adhesiveness was tested on Hep-2 cells, and specimens were processed for light, scanning and transmission electron microscopy. Adhesive capacity decreased over time with an increase in reduction percentages depending on NaCl concentrations. At 1·7% NaCl, the reduction was apparently temporary and adhesiveness rapidly recovered in revitalized bacteria, while 3·4%, 6·0% NaCl seemed to be detrimental. Normal, elongated and filamentous bacteria retained adhesiveness capability, although with reduced expression, while in spherical cells, this property seemed to be lost or dramatically reduced.

Conclusions

Our study shows that high osmolarity plays a significant role in adhesion inhibition, therefore having possible implications in the pathogenesis of the infections by Aer. hydrophila.

Significance and Impact of the Study

This study intends to give a contribution to a better understanding of the pathogenic role of this bacterium whose pathogenicity is still under debate.

Introduction

During their life cycle, bacteria are constantly exposed to a wide variety of stressful conditions; consequently, the ability to adapt to the changing and unfavourable environmental conditions is crucial for their survival. Bacteria have evolved mechanisms for sensing environmental conditions and respond by altering the pattern of gene expression with activation of genes, which allow them to survive and turning off those which are not necessary in a particular moment. As a consequence, the adaptation response may result in a modified expression of physiological and phenothypical characteristics. For example, authors refer that stress factors such as salinity, nutrient depletion, temperature, pH and atmosphere affect bacterial cell shape, suggesting that morphological changes are correlated to adaptive mechanisms that allow bacteria to prolong survival under adverse conditions (Mattick et al. 2000, 2003a,b; Everis and Betts 2001; Alonso et al. 2002; Shi and Xia 2003; Alterman et al. 2004; Jydegaard-Axelsen et al. 2005; Piuri et al. 2005; McMahon et al. 2007; Pianetti et al. 2009). Furthermore, several studies report that pathogenic bacteria may modulate their virulence properties in response to stress conditions, therefore influencing the outcome of the infectious process in the host. Among virulence factors adhesiveness to host cells is an early and critical step in bacterial infection, and it has been referred that its expression may change depending on the environment to which bacteria are exposed (Mihaljevic et al. 2007; Ellafi et al. 2009).

The aim of the present study was to evaluate, at the level of microscopical analysis, the adhesion properties of a strain of Aeromonas hydrophila isolated from fish following prolonged storage at refrigeration and room temperature in presence of different NaCl concentrations. This agent acts as an opportunistic as well as primary pathogen both in humans and in animals. Human infections range from gastroenteritis to extraintestinal diseases, such as wound infection, septicaemia and meningitidis (Janda and Abbott 2010). Aeromonas hydrophila is a common aquatic micro-organism frequently found in fresh water, groundwater, spring water, sea water, estuarine water, sediments, sewage-contaminated water, chlorinated and unchlorinated drinking water supplies, and even in bottled water (Fiorentini et al. 1998; Pianetti et al. 1998, 2004; Galindo and Chopra 2007). The bacterium has also been isolated from various foods such as vegetables, meat, ham, poultry, raw milk, fish and shellfish (Kirov 1997; Ottaviani et al. 2006; Janda and Abbott 2010). The distribution of Aer. hydrophila in different habitats underlines its ability to adapt to a wide variability of environmental conditions. In fact, it can grow or survive at temperatures ranging from <5 to 45°C (Kirov 1997), in vacuum packaged foods (Hudson et al. 1994), under 100% CO2 atmosphere (McMahon et al. 1998), in the presence of 0·34–1·02 mol l−1 sodium chloride concentrations (Delamare et al. 2000) and in poor nutrient conditions (Messi et al. 2002; Pianetti et al. 2005).

The pathogenicity of Aeromonas is attributed to a series of factors including cell structural component such as lipopolysaccharides (LPS), outer-membrane proteins (OMPs), pili and flagella, which function as adhesion structures and extracellular factors such as enzymes and toxins (Galindo and Chopra 2007). However, its pathogenic potential is still under debate. In fact, it has been evidenced that the expression of virulence factors in Aeromonas does not represent a stable state both at species and strain level and appears to be affected by environmental factors (Tso and Dooley 1995; Kirov 1997; Merino et al. 1998). Therefore, the present study on the influence of stressful conditions on Aer. hydrophila adhesion property intends to give a contribution to a better understanding of the pathogenic role of this problematic bacterium.

Materials and methods

Bacteria and microcosms

An Aer. hydrophila strain previously isolated from fish and identified by specific PCR assay (Ottaviani et al. 2011) was used in this study. nutrient broth (Oxoid, Milan, Italy) was used as the test medium. To adjust salt concentrations, it was modified by the addition of NaCl. Two series of three microcosms that contained different concentrations of NaCl (1·7%, 3·4% and 6·0%) were used.

Inoculation of microcosms

The Aer. hydrophila strain was regenerated in 5 ml of Tryptone Soya Broth (Oxoid) with 0·6% (w/v) yeast extract and 3% (w/v) Casamino Acids with overnight incubation at 28°C. One loopful of the culture was streaked on m-Aeromonas selective agar base (Havelaar; Biolife-Italiana, Milan, Italy) and incubated for 18–24 h at 28°C. Ten colonies were inoculated into 400 ml of Nutrient Broth and incubated at 28°C for 18–24 h with agitation at 150 rev min−1. The broth cultures were centrifuged at 1619 g for 30 min, and each pellet was resuspended in 10–15 ml of the same microcosm used for the experiments. The tests were performed in 2·5-l screw-cap Pyrex brown glass bottles. The Aer. hydrophila strain was inoculated into 2·3-l of each type of microcosm to obtain a bacterial concentration of 1·0 × 106 colony-forming units (CFU ml−1) as detected by OD and confirmed by plating. Each series of microcosms was incubated at 4 and 24°C, respectively. Furthermore, cultures were performed in unmodified Nutrient Broth and handled at the same conditions.

For bacterial revitalization, after a 188 day period, 10 ml of each microcosm and control culture was centrifuged at 1619 g for 30 min; the pellet was then resuspended in 10 ml of nutrient broth and inoculated into 490 ml of nutrient broth with incubation at the original temperature conditions for 15 h.

Adhesion assay

For the qualitative adhesion assay, bacteria were harvested by centrifugation (1619 g for 30 min) and the pellet was resuspended in 5 ml of Minimum Essential Medium (MEM) to obtain an absorbance (600 nm) of 0·07 (5·0 × 106 CFU ml−1). To verify bacterial viability, Live/Dead staining with Propidium Iodide was performed. Bacteria were then added to duplicate coverslip cultures of Hep-2 cells (IRCCS, Genova, Italy). The infected monolayers were incubated at 37°C for 90 min in air with 5% CO2. Non-adherent bacteria were removed by washing three times with phosphate-buffered saline (PBS). The cells were fixed with methanol/acetic acid (3 : 1) for 15 min and stained with 10% Giemsa for 1 h; the coverslips were mounted on glass slides and viewed by light microscopy.

The adhesive capacity was expressed as a percentage of cells with more than 10 bacteria per cell (Thornley et al. 1996). Bacteria which showed adhesiveness by optical microscopy were further analysed by electron microscopy. Bacterial adhesiveness was evaluated immediately before the inoculum in the microcosm and at intervals over a 188 day period as well as in the revitalized cultures. The assays were performed in triplicate.

Scanning electron microscopy

After washing, control-Hep-2 and Hep-2 with adhering bacteria, growing on coverslips, were fixed with 2·5% glutaraldehyde in mol l−1 phosphate buffer (pH 7·3) for 1 h. The monolayers were washed and postfixed with 1% OsO4 in the same buffer for 1 h. A progressive alcohol dehydration was performed, followed by specimen critical point drying. After mounting on conventional scanning electron microscopy (SEM) stubs by means of silver glue, slides were gold sputtered (Battistelli et al. 2005a). Observations were carried out with a Philips 515 scanning electron microscope.

Transmission electron microscopy

Normal and treated specimens were processed for transmission electron microscopy (TEM) according to conventional procedures (Battistelli et al. 2005b). Briefly, adherent cells were washed, immediately fixed with 2·5% glutaraldehyde in mol l−1 phosphate buffer (pH 7·3) for 5 min, gently scraped and centrifuged at 259 g. Pellets were additionally fixed for 1 h in 2·5% glutaraldehyde in 0·1 mol l−1 phosphate buffer. After a gentle washing, a postfixation was performed for 2 h in 1% OsO4 in the same buffer. Alcohol dehydration and araldite embedding were performed and thin sections, collected on nickel grids and stained with uranyl acetate and lead citrate were analysed with a Philips CM10 electron microscope.

Statistical analysis

The number of bacteria per Hep-2 cell was analysed by three-way anova model with respect to NaCl concentrations, temperature and time. Experiment wise significance level was fixed at 0·05.

Results

Values of adhesive bacteria were the mean of number of bacteria adhering to 30 Hep-2 cells. The tested Aer. hydrophila strain was initially highly adhesive, as detected by optical and SEM (14·2 ± 4·2 bacteria per cell) (Fig. 1b,d; Table 1). However, as reported in Table 1, adhesive capacity decreased over time at all culture conditions, but with not statistically significant differences (P > 0·05). In nutrient broth and in medium that contained 1·7% NaCl adhesiveness retained almost the initial values until day 21; then it gradually decreased reaching on day 188 values of 7·3 ± 6·1 and 6·5 ± 6·6 bacteria per cell at 4 and 24°C, respectively in Nutrient Broth and values of 4·7 ± 3·5 and 2·6 ± 1·7 at 4 and 24°C, respectively with 1·7% NaCl. In presence of 3·4% NaCl, number of adhesive cells at the end of the experiment were similar to those seen with 1·7% NaCl (4·6 ± 2·5 and 2·3 ± 2·6 at 4 and 24°C, respectively). However, in this case, a reduction of adhesiveness was observed starting from day 7 and was much more marked at 24°C (3·2 ± 2·7 bacteria per cell) than at 4°C (10·4 ± 3·2 bacteria per cell). Finally, at 6% NaCl, a drastic reduction was noted since day 3 (5·1 ± 2·6 bacteria per cell) at 24°C. At the end of the experiment, the values lowered to 1·8 ± 1·4 and 2·00 ± 1·9 bacteria per cell at 4 and 24°C, respectively.

Figure 1.

Light microscopy (a, b) and scanning electron microscopy (c, d) of control Hep-2 cells (a, c) and Hep-2 cells exposed to Aeromonas hydrophila strain. Aggregative adhesive patterns appear in b and d. (bar = 10 μm a and b).

Table 1. Hep-2 cell adhesion of Aeromonas hydrophila grown in different conditions
Time (days) T a Mean number of bacteriaPercentage of reductionMean number of bacteriaPercentage reductionMean number of bacteriaPercentage reductionMean number of bacteriaPercentage reduction
per Hep-2 cellin adhesionper Hep-2 cellin adhesionper Hep-2 cellin adhesionper Hep-2 cellin adhesion
Nutrient broth1·7% NaCl3·4% NaCl6·0% NaCl
  1. a

    Temperature (°C). Data are expressed as the means ± standard deviation.

0414·2 ± 4·2 14·2 ± 4·2 14·2 ± 4·2 14·2 ± 4·2 
2414·2 ± 4·2 14·2 ± 4·2 14·2 ± 4·2 14·2 ± 4·2 
3414·1 ± 3·90·7114·1 ± 3·70·7114·0 ± 4·11·4113·2 ± 3·57·05
2414·1 ± 4·30·7114·0 ± 3·71·4113·9 ± 3·92·125·1 ± 2·664·09
7414·0 ± 4·31·4113·9 ± 3·82·1210·4 ± 3·226·777·1 ± 2·850·0
2414·2 ± 4·50·013·6 ± 3·94·233·2 ± 2·777·473·0 ± 2·478·88
14413·6 ± 4·24·2312·9 ± 3·99·168·3 ± 2·541·552·8 ± 2·180·29
2413·2 ± 4·17·0513·5 ± 3·94·933·1 ± 2·578·171·8 ± 1·587·33
21413·3 ± 4·96·3412·8 ± 3·69·865·3 ± 4·162·682·5 ± 2·282·40
2412·9 ± 4·59·1612·7 ± 3·510·572·8 ± 1·780·291·7 ± 1·388·03
41410·2 ± 2·328·177·1 ± 3·050·05·2 ± 2·663·392·1 ± 1·885·22
247·5 ± 2·647·195·5 ± 2·261·272·4 ± 2·083·102·1 ± 1·685·22
6149·8 ± 2·330·996·3 ± 3·155·644·5 ± 3·068·312·7 ± 2·080·99
247·4 ± 2·547·895·5 ± 2·761·273·1 ± 2·678·171·6 ± 1·788·74
10148·5 ± 7·540·146·1 ± 3·257·054·8 ± 1·666·202·4 ± 1·983·10
246·7 ± 3·152·825·4 ± 3·861·983·2 ± 1·277·471·7 ± 1·288·03
13047·4 ± 2·947·895·1 ± 2·764·094·6 ± 2·867·611·7 ± 1·888·03
246·8 ± 2·752·123·2 ± 2·177·472·3 ± 1·783·811·8 ± 1·687·33
18847·3 ± 6·148·604·7 ± 3·566·914·6 ± 2·567·611·8 ± 1·487·33
246·5 ± 6·654·232·6 ± 1·781·702·3 ± 2·683·812·0 ± 1·985·92
Revitalised433·0 ± 4·9 40·0 ± 5·0 3·1 ± 1·778·173·1 ± 1·778·17
2431·3 ± 4·9 31·3 ± 4·9 6·9 ± 4·751·402·9 ± 1·579·58

When Aer. hydrophila was reinoculated in fresh Nutrient Broth, a remarkable increase in adhesiveness was shown by bacteria from control and 1·7% cultures at both temperatures. In fact, the number of adhesive bacteria increased to 33 ± 4·9 and 40 ± 5·0 bacteria per cell at 4°C in cultures from Nutrient Broth and from 1·7% NaCl, respectively, and to 31·3 ± 4·9 bacteria per cell at 24°C in both culture conditions. In the other experimental conditions used, the number of adhesive bacteria remained low (Table 1).

Scanning electron microscopy observation performed at the beginning of the experiment revealed the typical flat, elongated shape of Hep2 cells (Fig. 1c). Bacteria, at this time condition, appeared rod-shaped and showed aggregative adhesion (Fig. 1d).

For cultures in Nutrient Broth and in medium, which contained 1·7% NaCl stored at 4°C, normal-shaped bacteria were present although slightly elongated cells appeared throughout the experiment. With 3·4% NaCl, although in presence of normal-shaped bacteria, elongated and filamentous forms were evidenced since day 7 (Fig. 2a,b); elongated cells were present until day 188, starting from day 101 rare spherical forms were also seen (Fig. 2c). On day 188, very few adhering, moderately elongated, cells appeared. In this condition, ring-shaped cells were also present (Fig. 2d). With 6·0% NaCl, adhering bacteria were very scarce and appeared spherical. At 24°C, bacteria showed about the same morphological features as those grown at 4°C in all culture conditions.

Figure 2.

Scanning electron microscopy showing Hep-2 cells with adherent Aeromonas hydrophila. Normal-shaped bacteria at day 26 in 3·4% NaCl at 24°C appear in (a); filamentous forms at day 26 in 3·4% NaCl at 4°C are shown in (b); spherical bacteria at day 101 in 3·4% NaCl at 24°C are observable in (c); ring shaped bacteria appear at day 188 in 1·7% NaCl at 24°C (d). Normal and slightly elongated bacteria from 1·7% NaCl at 4°C appear after revitalization (e); pleomorphic bacteria from 3·4% at 24°C can be seen after revitalization (f).

The revitalization experiments showed normal or slightly elongated forms among bacteria adhering to Hep-2 cells, from cultures in Nutrient Broth and medium that contained 1·7% at both temperatures (Fig. 2e); in the other cases, together with slightly elongated forms, spherical cells were also present (Fig. 2f).

Transmission electron microscopy and SEM observations evidenced morphological changes in Hep-2 cells with adhering bacteria. Figure 3 shows control (Fig. 3a) and Hep-2 cells with adherent bacteria (Fig. 3b–h). Control cells appeared elongated (Fig. 3a), but after bacteria adhesion, they showed a rounded shape (Fig. 3b,c,e). The cells had numerous blebs and cytoplasm appeared richly vacuolated (Fig. 3b,d). Bacteria presented spherical or bacillary forms (Fig. 3c–f) and, occasionally, appeared internalized and vacuole surrounded (Fig. 3d). Cells with signs of necrosis were also revealed both by TEM and SEM observations, well recognizable by cell surface discontinuities (Fig. 3g) and progressive organelle destruction consequent to cytoplasm hydration (Fig. 3e,f,h).

Figure 3.

Transmission electron microscopy (a–f, h) and scanning electron microscopy (g) of control (a) and Aeromonas hydrophila-treated Hep-2 cells (b, d, e, f, g, h). Occasional cell rounding (b, c, e) appears after bacterial adhesion, which also generates cytoplasm vacuolization (v) and surface blebbing (b, d). Rarely bacteria appear internalized (d →). Cell necrosis patterns (n), recognizable as progressive organelle destruction and membrane discontinuities, can be also revealed (e–h) (bar = 1 μm).

Discussion

The present work focused on the adhesion properties of an Aer. hydrophila strain stored for 188 days at 4 and 24°C in presence of 1·7%, 3·4% and 6·0% NaCl concentrations. To our knowledge, there are no studies regarding the influence of a long exposure to subinhibiting NaCl concentrations on adhesiveness properties of Aer. hydrophila.

Aeromonads possess diverse surface structures, which function as adhesins; these include hemagglutinins associated with filamentous appendages or with OMPs as well as type IV pili, which have been purified from Aeromonas species isolated from diarrhoeic patients (Kirov and Sanderson 1995; Kirov et al. 1999; Rocha-De-Souza et al. 2001). Laboratory experiments evidenced that the ability of Aeromonas to adhere to Hep-2 cells is correlated with the enteropathogenicity of the genus (Kirov et al. 1995a). In addition, some studies have shown that adhesion to cell lines varies with bacterial growth conditions, suggesting that Aeromonas can express different adhesive abilities, depending on the environmental conditions (Kirov et al. 1995b; Aguilar et al. 1997).

Our results showed that a long exposure to suboptimal environmental conditions induced a general decrease in the adhesive capacity of the Aer. hydrophila strain with the beginning of the reduction apparently related to temperature and salt concentrations. In fact, in presence of less stressful conditions such Nutrient Broth and Nutrient Broth added with 1·7% NaCl, the decrease was noted from day 26, instead, in presence of increasing salt concentrations the decrease was anticipated at days 7 and 14 at 24 and 4°C, respectively with 3·4% NaCl, and at days 3 and 7 at 24 and 4°C, respectively with 6% NaCl. Afterwards, numbers of adhering bacteria gradually decreased until day 181 with reduction percentages apparently related not only to the prolonged temperature and osmotic stress, but also to nutrient depletion. In parallel, a viability study performed by flow cytometry and plate count technique (Pianetti et al. 2008) evidenced that, in spite of a long term halotolerance, there was a general increasing reduction in both viable cell number and CFU ml−1 according to the time, and NaCl concentrations. In fact, from initial values of 3·9 × 106 viability at the end of the experiment was reduced both in nutrient broth (7·0 × 105 and 3·7 × 104 at 4 and 24°C respectively) and in presence of 1·7% NaCl (2·8 × 106 and 3·4 × 104 at 4 and 24°C), respectively; this reduction increased similarly at salt concentrations of 3·4 and 6% (1·1 × 104 and 6·0–8·5 × 103 at 4 and 24°C respectively). However, in the initial phases, reduction of adhesiveness appeared not related to viability; in fact, this latter presented almost unvaried values (of the order of 106) until day 21.

Interestingly, in the revitalizing condition, adhesion capability highly recovered in bacteria from Nutrient Broth and Nutrient Broth that contained 1·7% NaCl cultures, while it kept impaired in those from 3·4 to 6·0% NaCl. Concomitantly, a general increase in viable cells (of 101–104 fold order) and CFU ml−1 (of 101–103 fold order) was seen irrespective to previous cultural conditions. Therefore, revitalization was not always associated with the reacquisition of adhesive properties. Observation of bacteria adhering to Hep-2 cells revealed the prevailing presence of normal and long cells, with an increase in elongation dependent on salt concentrations and incubation time. Adhering filamentous cells were also present, while spherical cells were very few. To better define the relationship between morphotype and adhesiveness, we compared the morphology of adhering cells with that of bacteria in suspension immediately before the adhesion experiments (Battistelli et al. 2007; Pianetti et al. 2009). In this case, the heterogenity of morphological forms dependent on salt concentrations was evident. In particular, bacteria analysed at the beginning of the experiment exhibited the normal rod-shaped morphology with polar flagellum. In control and 1·7% cultures, normal-shaped cells evolved to elongated and spherical forms during the experiment; the latter forms were prevalent in the last period as well as at 24°C. Filamentous, pili-like structures were observable on day 26 in the control culture at both temperatures, although more frequent at 4°C. In the presence of 3·4% NaCl, filamentous and spherical forms, with the first prevailing at 4°C and the latter at 24°C, were visible. Filamentous cells were appendage free, while rare pili appeared on spherical cells. In any case, normal-shaped bacteria were always present, though more frequent in control and in 1·7% NaCl culture. Finally, in 6·0% NaCl, greatly damaged cells could be seen at both temperatures all through the experiment. Revitalized cultures showed a large population of normal or slightly elongated cells, along with some filamentous and spherical forms. Pili-like structures could again be revealed in bacteria from Nutrient Broth and from 1·7% NaCl at 4°C. Although the presence of appendages on the cell surface of bacteria adhering to Hep-2 cells could not be revealed, the reduced adhesion observed could be associated with the loss of these appendages in suspended bacteria immediately before the adhesion experiment. On the other hand, the Hep-2 adhesion of appendage-free filamentous bacteria implies the role of other surface cell components such as OMP and lipopolysaccharide, as referred by several Authors (Merino et al. 1998; Rocha-De-Souza et al. 2001). Morphological changes can be considered adaptive mechanisms that allow bacteria to extend survival under adverse conditions. Our results suggest that harsh environmental conditions, such as nutrient limitation and osmotic stress, induce surface changes in Aer. hydrophila, which may affect not only its morphology, but also the adhesion process. Apparently normal, elongated and even filamentous forms can retain their adhesiveness capability although with reduced expression, while in spherical cells this property seems to be lost or dramatically reduced. Therefore, we can assume that the spherical state represents the ultimate response of bacteria to conditions of stress, which implies deep alterations in physical–chemical characteristics of the cell surface. Occasionally, adhering ring-shaped bacteria were also seen. To our knowledge, such forms have not been described in Aeromonas. Benaïssa et al. (1996) and Citterio et al. (2004) showed that Helicobacter pylori converted from bacillar to full coccoid form via an intermediate U-shaped form. Likewise, the observed ring-shaped cells could be regarded as a further folding of U-shaped cells that might represent a pre-VBNC state. To this regard, authors (Mary et al. 2002; Maalej et al. 2004; Pianetti et al. 2005) refer that Aer. hydrophila can enter into a VBNC state when grown in nutrient-poor conditions.

As also referred by other authors (Neves et al. 1994; Thornley et al. 1996), the tested Aer. hydrophila strain exhibited an aggregative adherence pattern. The adherence pattern was previously noted for members of Enterobacteriaceae which have been incriminated as important agents of diarrhoea (Jenkins et al. 2006).

Transmission electron microscope observation evidenced cytopathic and intracellular effects on Hep-2 cells with adhering bacteria that appears to be indicative of cytotoxic activity (Martins et al. 2002). This confirms the results of our previous study (Ottaviani et al. 2011) which demonstrated that the Aer. hydrophila strain was cytotoxic for VERO cells. Furthermore, in some cases, TEM revealed bacteria within the vacuoles, thus demonstrating both the ability to adhere to and invade epithelial cells. This is in agreement with other authors who report the ability of Aeromonas members to internalize (Lawson et al. 1985; Granum et al. 1998; Tan et al. 1998).

Our results are preliminary and further investigations are required to enhance understanding of the significance of our observations. In particular, it would be interesting to highlight the expression of the adhesion components, in the different morphotypes, which arise in response to environmental stress. The changes in adhesion ability observed in condition of moderate stress could be justified as an adaptation response to the surrounding environment through the modulation of the composition and structure of surface cell components. To this regard, studies evidenced that pH and osmolarity influenced the expression of type 1 pili in Escherichia coli (Schwan et al. 2002).

In conclusion, our study shows that, in the long term, osmolarity plays a significant role in adhesion inhibition, in particular at elevated salt concentrations irrespective of storage temperature, therefore having possible implications in the pathogenesis of the infections by Aer. hydrophila.

Acknowledgements

We thank F. Marinelli, Doctor in Statistical Science, Section of Occupational Medicine, Department Medical and Surgical Sciences, University of Bologna, Bologna, Italy, for his collaboration in the statistical analysis, Mr A. Valmori of IGM, CNR, Rizzoli Orthopedical Institute, Bologna, Italy is also thanked.

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