Biofilm formation and multidrug-resistant Aeromonas spp. from wild animals

Accepted Manuscript Title: Biofilm formation and multidrug resistant Aeromonas spp. from wild animals Authors: Carla Dias, Anabela Borges, Maria Jos´e Saavedra, Manuel Sim˜o es PII: DOI: Reference:

S2213-7165(17)30176-5 http://dx.doi.org/10.1016/j.jgar.2017.09.010 JGAR 499

To appear in: Received date: Revised date: Accepted date:

14-6-2017 12-7-2017 14-9-2017

Please cite this article as: Carla Dias, Anabela Borges, Maria Jos´e Saavedra, Manuel Sim˜o es, Biofilm formation and multidrug resistant Aeromonas spp.from wild animals (2010), http://dx.doi.org/10.1016/j.jgar.2017.09.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biofilm formation and multidrug resistant Aeromonas spp. from wild animals Subtitle: Aeromonas spp. biofilms and resistance

Carla DIASa,b,c Anabela BORGESa,c,d Maria José SAAVEDRAa,b,e Manuel SIMÕESC*

a

CECAV, Veterinary and Animal Science Research Center, University of Trás-os-Montes e Alto Douro,

5000-801 Vila Real, Portugal b

CITAB, Centre for the Research and Technology for Agro-Environment and Biological Sciences,

University of Trás-os-Montes e Alto Douro, 5000-801 Vila Real, Portugal c

LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, 4200-465

Porto, Portugal d

CIQUP, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do

Campo Alegre s/n, 4169-007 Porto, Portugal e

Department of Veterinary Sciences, School of Agriculture and Veterinary Science, University of Trás-os-

Montes e Alto Douro, 5000-801 Vila Real, Portugal

*Corresponding author: E-mail: [email protected]; Tel.: +351225081654; Fax: +351225081449

Highlights 

Wild animals are a source of multidrug resistant Aeromonas spp.



Strains of Aeromonas spp. have different biofilm formation abilities



All the strains are resistant to ciprofloxacin and multidrug resistant



Ciprofloxacin caused low to moderate biofilm removal

1

Abstract Objectives: The “One Health” concept recognizes that the health of humans, animals and the environment are interconnected. Therefore, the knowledge on the behavior of microorganisms from the most diverse environmental niches is important to prevent the emergence and dissemination of antimicrobial resistance. Wild animals are known to carry antimicrobial resistant microorganisms with potential public health impact. However, no data is available on the sessile behavior of bacteria from wild animals, even if antimicrobial resistance is amplified in biofilms. This study characterizes the antimicrobial susceptibility to ciprofloxacin, and the adhesion and biofilm formation abilities of 14 distinct Aeromonas spp. (eight A. salmonicida, three A. eucrenophila, two A. bestiarum and one A. veronii) isolated from wild animals and already characterized as resistant to β-lactam antibiotics. Methods: Minimal inhibitory concentration (MIC) of ciprofloxacin was determined according to Clinical and Laboratory Standards Institute guidelines. The biofilm formation assay was done according to a modified microtiter plate method. Bacterial surface hydrophobicity was assessed by sessile drop contact angles measurement. Results: All Aeromonas spp. strains were resistant to ciprofloxacin (minimum inhibitory concentrations between 6 and 60 µg/ml) and had hydrophilic surfaces (ranging from 2 and 37 mJ/m2). These strains were able to adhere and form biofilms with distinct magnitudes. Biofilm exposure to 10 times minimum inhibitory concentration of ciprofloxacin only caused low to moderate removal. Conclusion: This study shows that the strains tested are of potential public health concern and emphasize that wild animals are potential reservoirs of multidrug resistant strains. In fact, Aeromonas species are consistently considered opportunistic pathogens. Moreover,

2

bacterial ability to form biofilms increases antimicrobial resistance and the propensity to cause persistent infections.

Keywords: Aeromonas spp., biofilm formation, biofilm susceptibility, multidrug resistance, wild animals

1. INTRODUCTION The growth in the human population has prompted the encroachment of humans into new environments and resulted in environmental disruption of habitats, bringing humans and domestic animals into contact with wildlife. Close contact provides more opportunities for diseases to pass between animals and people [1]. Most of emerging infectious in people are spread from animals [2]. Those infections are problematic as they can reemerge after they are considered to be under control or eradicated. In many industrialized countries, diseases caused by antibiotic resistant bacteria are of particular importance for at-risk groups such as old, young, and immunocompromised individuals [3]. Surprisingly, new zoonotic diseases have been caused by opportunistic pathogens arising from wild animal reservoirs [4]. This clearly proposes that different ecosystems are inextricably connected. In this context, the “One Health” initiative proposes a global strategy developed for expanding interdisciplinary collaborations in all aspects of health care for humans, animals and the environment. The main aim is to achieve the best health [5]. Aeromonas form a complex genus of Gram-negative bacteria that can be found in most aquatic environments, including drinking water. Aeromonas species have received increasing attention as opportunistic pathogens [6]. They are important pathogens for fish and reptiles, and can occasionally cause disease in other animals and in humans [7]. Aeromonas-related infections are of particular concern in immunocompetent and

3

immunocompromised hosts [8], with systematic and localized manifestations like septicemia, wound infections, meningitis, endocarditis, and gastroenteritis [9]. The pathogenesis of Aeromonas species includes an extensive number of virulence determinants, many of them similar to virulence factors found in bacterial pathogens of humans [10]. Virulence mechanisms of Aeromonas are complex and poorly understood. Virulence-associated determinants include lipopolysaccharides, the S-layer outermembrane protein, pili and flagella, resistance to complete-mediated lysis and type III secretion system [7]. Besides their pathogenicity, a visible increase in resistance to antibiotics, including to β-lactams, has been observed not only for clinical strains but also for environmental strains [11]. Aeromonas species are capable to adhere on biotic or abiotic surfaces and form biofilms [12]. Bacterial biofilms are structured clusters of cells enclosed in a self-produced matrix of extracellular polymeric substance that are attached to a surface [13]. The biofilm lifestyle provides several advantages over growing planktonically, particularly increased resistance to antibiotics [14, 15]. This work was performed to characterize the antimicrobial susceptibility, adhesion and biofilm formation abilities of 14 diverse Aeromonas strains isolated from wild animals, providing insights into their virulence potential, environmental persistence and resilience.

2. METHODS 2.1 Aeromonas spp. strains The bacteria used in this work were obtained from faeces of wild animals (Table 1): four samples from Cervus elaphus (red deer) (A. salmonicida AS006C3c1,

AS006C3b1,

A. salmonicida

A. salmonicida AS006C3c2, A. salmonicida AS115A2GSP1, A. salmonicida AS117GSP2A,

A. salmonicida

AS117GSP2C1

owl) (A. salmonicida

and A. salmonicida

AS017A3a),

AS119GSP1C1),

one from Strix aluco (tawny

one from Sciurus vulgaris (red squirrel) (A. bestiarum 4

AS036A2a1B

and A. bestiarum

AS044A2a1(A),

AS036B1a1),

one from Colubridae (snake) (A. eucrenophila

A. eucrenophila AS044A2a1(B) and A. eucrenophila AS044A2a1(C)) and one sample

from Circaetus gallicus (short-toed snake eagle) (A. veronii

AS070GSP1),

as previously

described by Dias et al. [16].

2.2 Preparation of polystyrene as substratum Polystyrene (PS), a hydrophobic substratum, was selected for adhesion tests. In order to prepare PS, coupons with dimension of 1 cm × 1 cm were immersed in a solution of 5% (v/v) commercial detergent (Sonasol Pril, Henkel Ibérica S. A.) and in ultrapure water for 30 min with gentle shaking. To remove any remaining detergent, PS was rinsed five times in ultrapure water and immersed in ethanol at 96% (v/v) for 10 s. After being rinsed three time with ultrapure water, PS coupons were dried at 65 ºC for 3 h.

2.3 Determination of bacterial contact angles and surface hydrophobicity Contact angles were assessed according to Simões et al. [17] using a model OCA 15 Plus (DATAPHYSICS, Germany) video based optical contact angle measure instrument, allowing image acquisition and data analysis. Hydrophobicity was evaluated from contact angles values, as described by van Oss et al. [18-20]. The sessile drop contact angle with three different reference liquids, water, formamide and α-bromonaphthalene (Sigma, Portugal), were used to assess the surface tension of the bacteria. The surface tension components of the reference liquids were obtained from [21]. All assays were performed at room temperature and three independent experiments were performed for each strain.

2.4 Determination of minimal inhibitory concentration Minimal inhibitory concentration (MIC) of ciprofloxacin was determined according to the Clinical and Laboratory Standards Institute (CLSI [22] guidelines for broth 5

microdilution susceptibility testing of Aeromonas spp. Bacteria grown overnight (16-18 h incubation) in Mueller-Hinton Broth were adjusted to 1×106 CFU/ml and added to sterile 96-well polystyrene microtiter plates with ciprofloxacin. The final volume of each well was 200 µl and the antibiotic solution never exceed 10% of the well volume. The microtiter plates were incubated for 24 h at 30 ºC. The optical density (OD) of each well was measured at 640 nm using an automated microtiter plate reader (BIO-TEK, Model Synergy HT). The MIC corresponded to the lowest ciprofloxacin concentration at which no growth was detected. Water was used as negative control and all tests were performed in triplicate with three repeats.

2.5 Monolayer adhesion ability on polystyrene Coupons of PS, prepared as described previously, were placed in the bottom of 12-well (22 mm diameter each well) microtiter plates (polystyrene, Orange Scientific, USA) containing 2 ml of a bacterial culture (1×108 CFU/ml in Mueller-Hinton Broth). Initial adhesion to each coupon was allowed to occur by incubating the microtiter plates at 30 ºC for 2 h under 150 rpm agitation. Negative controls were obtained by placing coupons in MHB without bacteria. At the end of the assay coupons were withdrawn from the wells of the microtiter plates and immersed in 2 ml of sterile saline solution (NaCl, 0.85%) to remove the loosely attached bacteria. Afterwards the PS coupons were used for biomass quantification by crystal violet (CV) staining. All the assays were done in triplicate with three repeats.

2.6 Evaluation of biofilm formation The biofilm formation assay was done according to a modified microtiter plate method described by Stepanović et al. [23]. Briefly, 200 µl of a standardized bacterial culture (1×108 CFU/ml in MHB) was transferred into sixteen wells of a sterile 96-well microtiter 6

plate. The plate was incubated aerobically on a shaker at 150 rpm and 30 ºC, for 24, 48 and 72 h. For the 48 and 72 h old biofilms the medium was carefully discarded and replaced by fresh one on a daily basis. After each biofilm development period, the content of the wells was removed, and each well was washed three times with 250 μl of sterile saline solution to discard non-adhered bacteria. The microtiter plate was air dried for 30 min, and the remaining attached bacteria were analyzed in terms of biomass adhered on the surface of the microtiter plates. Wells with MHB were used as negative controls. All experiments were repeated three times.

2.6 Biomass quantification The biomass was quantified by crystal violet (CV) (Gram colour - staining set for microscopy, Merck, Portugal) staining according to Simões et al. [17]. For adhesion assays, PS coupons with adhered bacteria in the 12-well microtiter plates were removed, immersed in a new plate and the bacteria were fixed with 1 ml of ethanol 98% (v/v) (Vaz Pereira, Portugal). Ethanol was removed after 15 min of contact and the coupons were allowed to dry at room temperature. Afterwards the coupons were stained for 5 min with 600 µl of CV and washed with sterile saline solution, before being immersed in 1 ml of 33% (v/v) acetic acid (Merck, Portugal) to release and dissolve the stain. For biofilm mass quantification, the biofilms in the 96-well plates were fixed with 250 µl of 98% ethanol per well, for 15 min. Afterwards, the plates were air dried and the dye bound to the biofilm bacteria was resolubilized with 200 µl of 33% (v/v) glacial acetic acid. The optical density (OD) of each well was measured at 570 nm using an automated microtiter plate reader (BIO-TEK, Model Synergy HT).

2.7 Biofilm control 7

To determine whether ciprofloxacin had any effect on biofilm control, 24 h old biofilms formed in 96-well microtiter plates were exposed to 10 × MIC according to Simões et al. [17]. Twenty-four hours after exposure the biofilms were analyzed in terms of mass reduction by CV staining. Biofilm removal was given by: % BR =

ODn−ODa ODn

× 100, where

% BR is the percentage of biofilm removal, ODn is the OD570 nm value of biofilm nonexposed to ciprofloxacin and ODa is de OD570

nm

value for biofilm exposed to the

antibiotic. The action of ciprofloxacin on biofilm removal was classified according to Lemos et al. [24]: Removal < 25% - low efficacy; Removal < 60% - moderate efficacy; 60 ≤ Removal < 90% - high efficacy; 90 ≤ Removal ≤ 100% - excellent efficacy.

2.8 Statistical analysis Data were analyzed by analysis of variance (ANOVA) using the statistical program SPSS version 22.0 (Statistical Package for the Social Sciences). Statistical calculations were based on a confidence level ≥ 95% (P < 0.05 was considered statistically significant).

3. RESULTS 3.1 Hydrophobicity 𝑇𝑂𝑇 The surfaces of all Aeromonas strains are hydrophilic, ΔG𝑏𝑤𝑏 ˃ 0 mJ/m2 (Table 1).

However, the hydrophobicity values were significantly different between some of the strains. Strain A. eucrenophila

AS044A2a1(C)

while A. salmonicida

had the lowest value. This difference in hydrophobicity

AS006C3c2

had the highest surface hydrophobicity value

was statistically significant (P < 0.05). In the A. salmonicida group, three strains isolated from the same animal species, Cervus elaphus, showed statistically significant differences in their hydrophobicity values (P < 0.05). The six remaining A. salmonicida strains

8

(AS006C3c2,

AS017A3a,

AS115A2GSP1,

AS117GSP2A,

AS117GSP2C1

and

AS119GSP1C1)

had similar

hydrophobicity values that were not different from each other (P > 0.05). Regarding the remaining physicochemical parameters, the apolar component (γLW) was in the range of 40 to 42 mJ/m2 for A. salmonicida strains, with the exception of A. salmonicida AS006C3b1 (γLW = 33 mJ/m2). However, this strain had the highest surface polar component (γAB = 24 mJ/m2). A. salmonicida AS017A3a had the lowest value for the surface polar component (γAB = 5.8 mJ/m2). A. bestiarum AS036A2a1B had the lowest value of apolar component while A. salmonicida

AS119GSP1C1

had the highest value. All the bacteria had

predominantly electron donor surfaces (high γ- values), with low electron acceptor ability (γ+). Furthermore, linear regression showed a strong correlation (R2 = 0.98) between the electron donor parameter and the bacterial surface hydrophobicity (Fig. 1).

3.2 Ciprofloxacin minimum inhibitory concentration The MIC of ciprofloxacin was assessed for the diverse Aeromonas strains (Table 2). The MICs for eight of those strains was 6 µg/ml. MICs for A. salmonicida salmonicida

AS017A3a,

A. salmonicida

AS006C3b1

and A. salmonicida

AS006C3c1

AS006C3c2,

A.

were 32, 36,

48 and 60 µg/ml, respectively.

3.3 Monolayer adhesion ability Initial bacterial adhesion to PS was also evaluated using a microtiter plate assay and CV staining for biomass quantification (Fig. 2). The diverse strains showed varying levels of adhesion. Of all strains, 57% (8/14) were classified as moderately adherent to PS. A. bestiarum

AS036B1a1

and A. salmonicida

AS006C3c2

had the highest and lowest adhesion

abilities, respectively (Table 3).

3.4 Biofilm formation 9

Fig. 3 shows the mass of biofilms with different ages (24, 48 and 72 h). The results show that all strains were able to form biofilms, however, they had statistically distinct productivities (P < 0.05). A. salmonicida AS006C3c1 produced the highest biomass amount for 72 h, A. salmonicida AS006C3b1 for 48 h and A. veronii AS070GSP1 for 24 h sampling times. A. eucrenophila AS044A2a1(B), A. salmonicida AS017A3a and A. salmonicida AS006C3c2 produced the lowest biomass amount for 24, 48 and 72 h sampling times, respectively. The diverse strains were classified in terms of biofilm productivity as weak, moderate or strong biofilm producers (Table 3). A. salmonicida AS006C3b1 and A. salmonicida AS006C3c1 showed a strong ability of biofilm formation at 48 and 72 h sampling times and A. salmonicida AS115A2GSP1

showed a strong ability of biofilm formation at 72 h sampling time. A.

salmonicida

AS006C3c2

and A. salmonicida

AS017A3a

showed a weak ability of biofilm

formation for all sampling times. A. veronii AS070GSP1 showed variability in the ability of biofilm formation, showing a moderate ability at 24 h, a weak ability at 48 h and a moderate ability again at 72 h. A. eucrenophila

showed a weak ability of

AS044A2a1(A)

biofilm formation at 24 h and moderate ability at 48 and 72 h.

3.5 Ciprofloxacin on biofilm removal The ability of ciprofloxacin to control 24 h old biofilms was analyzed in terms of mass removal (Fig. 4). Moderate removal (> 25%) was found for nine strains (A. salmonicida AS006C3b2,

A. salmonicida

AS006C3c1,

A. salmonicida

AS006C3c2,

A. salmonicida

AS017A3a,

A.

bestiarum AS036A2a1B, A. bestiarum AS036B1a1, A. eucrenophila AS044A2a1(A), A. eucrenophila AS044A2a1(B)

and A. veronii

AS070GSP1).

The most significant removal, with statistical

significance compared to other strains (P < 0.05), was found for biofilms of A. salmonicida AS006C3c2 (52%), A. bestiarum AS036B1a1 (56%) and A. eucrenophila AS044A2a1(B) (52%). Ciprofloxacin had low effects (removal < 25%) on the removal of biofilms formed by the other strains. In fact, no mass removal was observed for biofilms formed by A. 10

eucrenophila

AS044A2a1(C),

A. salmonicida

AS117GSP2A,

A. salmonicida

AS117GSP2C1

and A.

salmonicida AS119GSP1C1.

4. DISCUSSION In the present study, 14 different Aeromonas strains were isolated from five distinct wild animal species. These strains were already found to be resistant to β-lactam antibiotics, therefore representing a potential risk to public health [16]. The treatment of Aeromonas infections can be done with different classes of antibiotics, particularly β-lactam, chloramphenicol and tetracycline antibiotics [25]. However, an increase in the resistance to those antibiotics among disease-causing Aeromonas species has been reported. In the present study, the susceptibility of the Aeromonas strains to ciprofloxacin was assessed. This antibiotic was selected due to its current use for the treatment of human and animal bacterial infections [26]. The MIC of ciprofloxacin varied from 6 to 60 µg/ml. According to the MIC breakpoint of ≥ 4 µg/ml for ciprofloxacin-resistant Aeromonas species [37], all strains tested in this study are considered resistant to ciprofloxacin. These results together with the resistance patterns of previous antibiotic susceptibility tests [31] indicate that all strains under study are resistant to more than two antibiotics. Previous studies [27] reported quinoloneresistant strains of Aeromonas recovered from humans, freshwater and eels. Of particular concern was the detection of A. caviae and A. veronii strains, isolated from stools of patients with gastrointestinal symptoms, and resistant to nalidixic acid [27]. To our knowledge this is the first report showing multidrug-resistant Aeromonas isolated from wild animals. The Aeromonas strains were characterized based on their surface properties and their abilities for adhesion and biofilm formation. It is widely accepted that bacterial surface hydrophobicity is a key factor in the adhesion process and further biofilm development 11

[28]. In this study, all 14 strains had hydrophilic surfaces. Although, they belonged to four different Aeromonas species, no species-specific differences in their surface hydrophobicity were found. Strains isolated from a particular animal did not show a characteristic pattern of hydrophobicity. Moreover, no correlation was found between surface hydrophobicity and the magnitude of adhesion and biofilm formation. Similarly, no correlations were observed by comparing the remaining physicochemical surface properties (γLW, γAB, γ+ and γ-) with bacterial adhesion and biofilm formation. Bacterial adhesion to host tissues is an essential initial step in the infection process. Bacteria isolated from different niches typically exhibit different abilities to adhere on the substratum and to form biofilms. Such distinct abilities depend not only on the surface characteristics and the environment surrounding the microorganism (nutrients, ionic strength, pH and temperature) but also on their phenotype and genotype [29]. Several studies indicate that Aeromonas spp. are able to adhere on different surfaces such as stainless steel, copper, polybutylene and rubber [28, 30]. In this study, the tested Aeromonas strains adhered on PS, however, they exhibited different adhesive strengths regardless of their animal source or bacterial species. In order to evaluate whether the initial bacteria-PS binding event was determinant for subsequent biofilm development, the ability of the isolated strains to form biofilms was evaluated. PS microtiter plates were used in this study as standard bioreactor system for adhesion and biofilm formation. This type of device was used in similar studies with bacteria from different environments providing reliable comparative data [17, 31]. No clear relationship was found between the amount of biofilm formed and the initial monolayer adhesion extent. For instance, strains A. salmonicida AS006C3b1, A. eucrenophila AS044A2a1(A),

A. eucrenophila

AS044A2a1(C),

A. salmonicida

AS117GSP2C1,

had weak monolayer adhesion ability, however, these strains were classified

AS117GSP2A

and A. salmonicida

12

as moderate biofilm formers after 48 h of growth in PS microtiter plates. These results suggest that biofilm formation is more likely to be influenced by cell-cell aggregation and interactions than by the initial adhesion of the strains to the substratum [32]. The data from the present study demonstrates that each strain had a different ability to form biofilms, even under the same experimental conditions. A similar conclusion was obtained by Chenia and Duma [33] when studying the biofilm formation ability of Aeromonas strains isolated from diverse freshwater fish species and from sea water. In a recent study Ormanci and Yucel [34] reported biofilm formation by diverse Aeromonas species (food and environmental isolates) during 24 h and found OD570nm ranging from 0.090 and 1.255. These values clearly propose that the Aeromonas species tested in the present study had higher biofilm productivity. However, it is important to refer that different growth media were used for biofilm formation: MHB – selected as standard medium for antibiotic testing [22] and Tryptic Soy Broth-TSB or Brain-Heart InfusionBHI [34]. In fact, Ormanci and Yucel [34] proposed that different growth media, due to their diverse ingredients, had distinct effects on biofilm formation, with less-rich growth medium (TSB) favouring biofilm formation. Dewanti and Wong [35] also found that Escherichia coli developed biofilm faster and with a higher cell density when grown in low-nutrient media. Hood and Zottola [36] demonstrated that Salmonella typhimurium produce more biofilm in nutrient-limited media, while Listeria monocytogenes produce more biofilm in nutrient-rich medium. Moreover, it needs to be taken into account that bacterial adhesion to surfaces and biofilm formation is dependent on the specific strain and is not a general characteristic of a bacterial species or serotype [37, 38]. Biofilm formation ability was strongly dependent on the time, i.e. longer biofilm formation periods increased biomass productivity, except for A. salmonicida AS017A3a and A. salmonicida AS006C3c1 strains. After 24 hours of incubation the majority of strains (86%)

13

had weak ability to form biofilm. Only strain A. veronii

AS070GSP1

had moderate biofilm

formation ability. Results of Igbinosa et al. [39] showed that 53% of Aeromonas strains isolated from two rivers were weak biofilm producers, 29% were strong producers while 16% demonstrated moderate ability to form biofilms. Odeyemi et al. [40] reported that A. hydrophila isolated from estuaries were able to form biofilms, even if most of the strains were weak biofilm producers. In the present study, using four different Aeromonas species, a similar behavior was observed. The majority of strains (except A. veronii AS070GSP1,

classified as moderate biofilm producer) had weak biofilm producing ability at

24 h sampling time. Increasing the biofilm formation period also increased biomass production, for most of the cases, with the exception of A. salmonicida salmonicida

AS017A3a

and A. veronii

AS070GSP1.

AS006C3c2,

A.

The presence of Aeromonas spp. with

biofilm-producing potential is of great concern to public health because these organisms can harbour other pathogens [9]. Studies of Kirov et al. [30] comparing in-vitro biofilm assays and biofilm formation on human tissues, reported that the ability of Aeromonas strains to form biofilms further reveals their pathogen status. Due to the recognized higher resistance of sessile communities when compared with their planktonic counterparts [17], biofilms were exposed to ciprofloxacin with 10 times above the MIC. This treatment caused biofilm mass reductions from 0 to 58%. Ciprofloxacin was previously tested in biofilm control, providing inconclusive results. Lewis [41] proposed that the majority of biofilm bacteria were killed within a ciprofloxacin concentration ≤ 5 µg/ml. In another study, Walters et al. [42] reported the effect of ciprofloxacin (1.0 µg/ml) against Pseudomonas aeruginosa biofilms and observed that the antibiotic penetrated biofilms but failed to kill the bacteria. Later, Lewis [43] observed that fluoroquinolones are effective in preventing biofilm formation, but once the biofilm community is mature the antimicrobial agent is restricted in its ability to diffuse through

14

the biopellicle and reach the target site. This is one possible explanation for the reduced effects of ciprofloxacin in biofilm removal found in this study. In fact, biofilms are heterogeneous and complex structures of microorganisms presenting sophisticated singular and collective behaviours which allows the colonizing microorganisms to survive in hostile environments [32]. In conclusion, this work demonstrates that Aeromonas strains isolated from wild animals have the ability to adhere on PS and form biofilms. Initial adhesion and biofilm formation on abiotic surfaces are important virulence mechanisms of bacteria to facilitate survival within host cells and in adverse environmental conditions. These characteristics are strain-dependent and do not represent a species-specific phenotype or correspond to the source of bacterial strains. Moreover, there is an extensive range of variation in adhesion and biofilm formation among the Aeromonas strains. The 14 strains were resistant to ciprofloxacin and multidrug resistant. Moreover, the use of ciprofloxacin with 10 times above the MIC was not effective in biofilm removal. Therefore, the overall results propose that the strains tested are of potential public health concern. In fact, most Aeromonas strains are consistently considered opportunistic pathogens and the ability to form biofilms will increase their antimicrobial resistance, and their potential to cause persistent infections.

Declarations Funding: This work was financially supported by: This work was financially supported by: Project POCI-01-0145-FEDER-006939 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia; by COMPETE, FCT/MEC (PIDDAC) and FEDER through Project Phytodisinfectants - PTDC/DTP-SAP/1078/2012 (COMPETE: FCOMP-01-0124-FEDER-028765) and the

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PhD grant awarded to Carla Dias (SFRH/BD/90120/2012) and the Post-Doc grant awarded to Anabela Borges (SFRH/BPD/98684/2013).

Competing Interests: Not applicable Ethical Approval: Not applicable

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[20] van Oss CJ, Chaudhury MK, Good RJ. Estimation of the polar parameters of the surface tension of liquids by contact angle measurements on gels. J Colloid Interface Sci. 1989;28:313-9. [21] Janczuk B, Chibowski E, Bruque JM, Kerkeb ML, Caballero FG. One the consistency of surface free energy components as calculated from contact angle of different liquids: an application to the cholesterol surface. J Colloid Interface Sci. 1993;159:421-8. [22] CLSI. Performance standards for antimicrobial susceptibility testing; 24th informational supplement. M100-S24. Clinical and Laboratory Standards Institute, Wayne. 2014. [23] Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Meth. 2000;40:175-9. [24] Lemos M, Borges A, Teodósio J, Araújo P, Mergulhão FJ, Melo LF, et al. The effects of ferulic and salycilic acids on Bacillus cereus and Pseudomonas fluorescens single- and dual-species biofilms. Int Biodeterior Biodegradation. 2014;86:42-51. [25] Ko WC, Chiang SR, Lee HC, Tang HJ, Wang YY, Chuang YC. In vitro and in vivo activities of fluoroquinolones against Aeromonas hydrophila. Antimicrob Agents Chemother. 2003;47:2217-22. [26] Igbinosa IH, Igbinosa EO, Okoh AI. Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle. Environ Sci Pollut Res Int. 2015;22:17596-605. [27] Arias A, Seral C, Gude MJ, Castillo FJ. Molecular mechanisms of quinolone resistance in clinical isolates of Aeromonas caviae and Aeromonas veronii bv. sobria. Int Microbiol. 2010;13:135-41.

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[28] Kregiel D. Adhesion of Aeromonas hydrophila to glass surfaces modified with organosilanes. Food Technol Biotechnol. 2013;51:345-51. [29] Thomas WE, Trintchina E, Forero M, Vogel V, Sokurenko EV. Bacterial adhesion to target cells enhanced by shear force. Cell. 2002;109:913-23. [30] Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun. 2004;72:1939-45. [31] Monte J, Abreu AC, Borges A, Simões LC, Simões M. Antimicrobial activity of selected phytochemicals against Escherichia coli and Staphylococcus aureus and their biofilms. Pathogens. 2014;3:473-98. [32] Alves MM, Pereira MA, Novais JM, Polanco FF, M. M. A new device to select microcarriers for biomass immobilization: application to an anaerobic consortium. Water Environ Res. 1999;71:209-17. [33] Chenia HY, Duma S. Characterization of virulence, cell surface characteristics and biofilm-forming ability of Aeromonas spp. isolates from fish and sea water. J Fish Dis. 2016:1-12. [34] Ormanci S, Yucel N. Biofilm formation on polystyrene and glass surface by Aeromonas species isolated from different sources. J Food Process Preserv. 2017:1-8. [35] Dewanti R, Wong AC. Influence of culture conditions on biofilm formation by Escherichia coli O157:H7. Int J Food Microbiol. 1995;26:147-64. [36] Hood SK, Zottola EA. Adherence to stainless steel by foodborne microorganisms during growth in model food systems. Int J Food Microbiol. 1997;37:145-53. [37] Santiago NI, Zipf A, Bhunia AK. Influence of temperature and growth phase on expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes. Appl Environ Microb. 1999;65:2765-9.

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[38] Elhariry HM. Biofilm Formation by Aeromonas hydrophila on Green-Leafy Vegetables: Cabbage and Lettuce. Foodborne Pathog Dis. 2011;8:125-31. [39] Igbinosa IH, Chigor VN, Igbinosa EO, Obi LC, Okoh AI. Antibiogram, adhesive characteristics, and incidence of class 1 integron in Aeromonas species isolated from two South African rivers. BioMed Res Int. 2013;2013:127570. [40] Odeyemi OA, Asmat A, Usup G. Antibiotics resistance and putative virulence factors of Aeromonas hydrophila isolated from estuary. Microbiol Biotechnol Food Sci. 2012;1:1339-57. [41] Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45:9991007. [42] Walters MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS. Contributions of antibiotic penetration, oxygen limitation, and metabolic activity to tolerance of Pseudomonas aeruginosa biofilm to ciprofloxacin and tobramycin. Antimicrob Agents Chemother. 2003;47:317-23. [43] Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol. 2007;5:48-56.

21

Figure legends Fig. 1. Correlation between the electron donor parameter (γ-) and the bacterial surface TOT hydrophobicity (∆𝐺bwb ). A strong correlation (R2 = 0.98) between the two parameters

was obtained.

Fig. 2. Values of OD570 nm as a measure of monolayer bacterial adhesion on PS for 2 h, showing the varying levels of adhesion of the diverse strains, with A. bestiarum AS036B1a1 and A. salmonicida

AS006C3c2 having

the highest and lowest adhesion abilities, respectively. The

mean ± SD of three independent experiments are illustrated.

Fig. 3. Values of OD570nm as a measure of biomass of 24 h ( ), 48 h ( ) and 72 h ( ) hours old biofilms. All strains were able to form biofilms. A. salmonicida produced the highest biomass amount for 72 h, A. salmonicida

AS006C3b1

AS006C3c1

for 48 h and A.

veronii AS070GSP1 for 24 h. A. eucrenophila AS044A2a1(B) (24 h), A. salmonicida AS017A3a (48 h) and A. salmonicida AS006C3c2 (72 h) produced the lowest biomass amounts. The mean ± SD of three independent experiments are illustrated.

Fig. 4. Percentage of biofilm reduction for the Aeromonas strains exposed to 10 × MIC of ciprofloxacin for 24 h. The most significant removal was found for biofilms of A. salmonicida

AS006C3c2,

A. bestiarum

AS036B1a1

and A. eucrenophila

AS044A2a1(B).

* - Means

no biofilm removal. Mean values ± SD for at least three replicates are illustrated.

22

Fig. 1.

23

A . s a lm o n ic id a A S119G SP 1C1

A . s a lm o n ic id a A S117G SP 2C1

A . s a lm o n ic id a AS117GSP2A

A . s a lm o n ic id a AS115A2GSP1

A . v e r o n ii AS070GSP1

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (C)

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (B )

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (A )

A . b e s tia r u m AS036B1a1

A . b e s tia r u m AS036A2a1B

A . s a lm o n ic id a AS017A3a

A . s a lm o n ic id a A S 0 0 6 C3 c2

A . s a lm o n ic id a A S 0 0 6 C3 c1

A . s a lm o n ic id a A S006C3b1

0.

0

0.

1

0.

2

0.

OD

3

0.

4

0.

5

570 nm

Fig. 2.

24

A . s a lm o n ic id a A S119G SP 1C1

A . s a lm o n ic id a A S117G SP 2C1

A . s a lm o n ic id a AS117GSP2A

A . s a lm o n ic id a AS115A2GSP1

A . v e r o n ii AS070GSP1

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (C)

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (B )

A . e u c r e n o p h ila A S 0 4 4 A 2 a 1 (A )

A . b e s tia r u m AS036B1a1

A . b e s tia r u m AS036A2a1B

A . s a lm o n ic id a AS017A3a

A . s a lm o n ic id a A S 0 0 6 C3 c2

A . s a lm o n ic id a A S 0 0 6 C3 c1

A . s a lm o n ic id a

0 6.

5

5 5.

6.

0 5.

0 3.

5

5 2.

4.

0 2.

0

5 1.

4.

0 1.

5

5 0.

OD

3.

0 0.

A S006C3b1

570 nm

Fig. 3.

25

A

17

G

S

.s al

1

on

1

on

SP

SP

m

2G

lm

0G

S1

7G

G

SP

1

1C

1

on

2C

lm

SP

.s a 19

A

ic

ic

ic

id

id

id

id

a

a

a

a

i

la

la

ni

hi

ic

m

ila

hi

ro

op )

. s P2A al m S1 on 1

A

S1

A

A

.s a 15

A

C

.v e

1(

A

2a

S7

A

A

44

)

en

B

op

)

en 1(

cr

2a

A

ru

ph

ia

* A

A

1(

1

no

1a

re

cr

2a

.e u

44

S1

A

A

.e u

44

B

m

a

a

0

A

36

1B

id

id

ru

ic

a

a

id

id

ic

tia

on

st

2a

.b e

A

.e uc

S0

A

S0

A

S0

A

A

A

A

S0

3a

lm

A

2

.b es

36

A

S0

A

17

3c

on

1

ic

ic

on 1

on

m

3c

C

.s a

06

S0

A

A

A

A

C

.s al

06

S0

A

S0

A

A

3b

lm

m

C

.s al

06

.s a

S0

A

A

A

B io film R e d u c tio n (% )

60

50

40

30

20

10

* * *

Figure 4.

26

Table 1. Contact angles (in degrees) with water (θW), formamide (θF), αbromonaphthalene (θB), surface tension parameters (γLW, γAB, γ+, γ-), free energy of 𝑇𝑂𝑇 interaction (∆𝐺𝑏𝑤𝑏 ) of the Aeromonas strains (b) when immersed in water (w). Values

are mean ± SD of three independent experiments Contact angle (º) θW Source wild animal

θB

θF

Surface tension parameters (mJ/m2) γLW γAB γ + γ-

Hydrophobicity (mJ/m2) ∆𝑮𝑻𝑶𝑻 𝒃𝒘𝒃

Strains A. salmonicida AS006C3b1

20±2 44±3 14±2 33±0 24±0 3±0

49±0 24±1

A. salmonicida AS006C3c1

18±1 27±2 20±2 40±0 14±0 1±0

54±0 32±0

A. salmonicida AS006C3c2

39±6 21±3 29±4 41±0 10±1 0.3±0 37±0 2±1

A. salmonicida AS115A2GSP1

20±2 46±1 19±1 42±0 6±0

A. salmonicida AS117GSP2A

14±1 39±1 14±1 40±0 13±0 1±0

A. salmonicida AS117GSP2C1

41±2 19±1 34±2 42±0 6±0

0.3±0 38±0 14±0

A. salmonicida AS119GSP1C1 Circaetus gallicus (short-toed snake A. veronii AS070GSP1 eagle)

41±1 17±2 30±3 42±0 8±0

0.5±0 36±0 9±0

Cervus (red deer)

elaphus

Colubridae (snake) Sciurus vulgaris (red squirrel) Strix aluco (tawny owl) 2

41±1 16±1

40±2 17±3 34±2 36±0 18±0 2±0

53±0 31±0

A. eucrenophila AS044A2a1(A) 27±2 37±2 32±1 36±0 12±0 1±0

53±0 34±0

A. eucrenophila AS044A2a1(B) 23±1 35±1 27±1 37±0 14±0 1±0

54±0 34±1

A. eucrenophila AS044A2a1(C) 22±7 42±1 31±2 34±0 15±0 1±0

56±1 37±1

A. bestiarum AS036A2a1B

20±2 46±1 19±1 32±0 23±0 3±0

51±0 27±1

A. bestiarum AS036B1a1

14±1 39±1 14±1 35±0 21±0 2±0

53±0 30±1

A. salmonicida AS017A3a

43±3 20±1 35±1 42±0 6 ±1

0.2±0 36±1 10±1

83±3 71±2 28±1 39

0.0

PS 𝑇𝑂𝑇 ∆𝐺𝑏𝑤𝑏

0.2±0 39±1 15±1

< 0 mJ/m - hydrophobic surface;

𝑇𝑂𝑇 ∆𝐺𝑏𝑤𝑏

0.0

9.9

-44

2

> 0 mJ/m - hydrophilic surface.

27

Table 2. Minimum inhibitory concentrations of ciprofloxacin for different Aeromonas strains Source wild animal

Cervus (red deer)

elaphus

Circaetus gallicus (short-toed snake eagle) Colubridae (snake) Sciurus vulgaris (red squirrel) Strix aluco (tawny owl)

Species

MIC (µg/ml)

A. salmonicida AS006C3b1

48

A. salmonicida AS006C3c1

60

A. salmonicida AS006C3c2

32

A. salmonicida AS115A2GSP1

6

A. salmonicida AS117GSP2A

6

A. salmonicida AS117GSP2C1

6

A. salmonicida AS119GSP1C1

6

A. veronii AS070GSP1

6

A. eucrenophila AS044A2a1(A)

6

A. eucrenophila AS044A2a1(B)

6

A. eucrenophila AS044A2a1(C)

6

A. bestiarum AS036A2a1B

24

A. bestiarum AS036B1a1

24

A. salmonicida AS017A3a

36

28

Table 3. Adhesion and biofilm formation ability of Aeromonas strains to PS according to the classification proposed by Stepanović et al. [23] Source wild animal

Cervus (red deer)

elaphus

Circaetus gallicus (short-toed snake eagle) Colubridae (snake) Sciurus vulgaris (red squirrel) Strix aluco (tawny owl)

Species

Adhesion

Biofilm 24 h

48 h

72 h

A. salmonicida AS006C3b1

+

+

+++

+++

A. salmonicida AS006C3c1

++

+

+++

+++

A. salmonicida AS006C3c2

+

+

+

+

A. salmonicida AS115A2GSP1

++

+

++

+++

A. salmonicida AS117GSP2A

+

+

++

++

A. salmonicida AS117GSP2C1

+

+

+

++

A. salmonicida AS119GSP1C1

++

+

++

++

A. veronii AS070GSP1

++

++

+

++

A. eucrenophila AS044A2a1(A) +

+

++

++

A. eucrenophila AS044A2a1(B) ++

+

+

++

A. eucrenophila AS044A2a1(C) +

+

+

++

A. bestiarum AS036A2a1B

++

+

++

++

A. bestiarum AS036B1a1

++

+

++

++

A. salmonicida AS017A3a

++

+

+

+

(0) OD≤2×ODc - non-adherent/non-biofilm producer; (+) ODc
29