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European Journal of Applied Sciences – Vol. 12, No. 1

Publication Date: February 25, 2024

DOI:10.14738/aivp.121.16462

Bino, E., Kandričáková, A., & Lauková, A. (2024). Biofilm Formation in Coagulase-Negative Staphylococci from Various Animals.

European Journal of Applied Sciences, Vol - 12(1). 317-324.

Services for Science and Education – United Kingdom

Biofilm Formation in Coagulase-Negative Staphylococci from

Various Animals

Eva Bino

Centre of Biosciences of the Slovak Academy of Sciences

Institute of Animal Physiology, Šoltésovej 4-6, Košice 040 01, Slovakia

Anna Kandričáková

Centre of Biosciences of the Slovak Academy of Sciences

Institute of Animal Physiology, Šoltésovej 4-6, Košice 040 01, Slovakia

Andrea Lauková

Centre of Biosciences of the Slovak Academy of Sciences

Institute of Animal Physiology, Šoltésovej 4-6, Košice 040 01, Slovakia

ABSTRACT

Bacteria show a distinct tendency to "adhere" to various surfaces. This is precisely

why many microorganisms occur in the environment in the form of a biofilm and

not in a planktonic form. Biofilm formation has been demonstrated in several

bacterial species, and hence in staphylococci. Previous studies regarding the

staphylococci related to human strains. Antibiotic resistance is currently a problem

all over the world, and the formation of biofilm can also affect it, since bacteria that

grow in the form of biofilm are much more resistant. The aim of this study was

testing biofilm-forming ability in various staphylococci from different animals. One

hundred (100) faecal staphylococci from 407 animals were tested. Biofilm

formation tested on Congo red agar was confirmed after 72 hours in 81

staphylococci, in 19 strains biofilm was not confirmed on this medium. Using tube

method correlation in most cases with the results on Congo red agar was found.

Microtiter quantitative plate assay assessed biofilm production in 59 staphylococci

out of 100 tested. In a percentage, 96.29% strains from faeces of domestic animals

formed biofilm. In the species Staphylococcus vitulinus (14), S. pasteuri (1), S. sciuri

(2), S. saprophyticus (1), and S. caprae (1) was biofilm-forming ability detected only

using plate assay. To know biofilm-forming ability in huge target of coagulase- negative staphylococci from various animal species is original contribution to

biofilm studies.

Keywords: biofilm, staphylococci, animals

INTRODUCTION

Staphylococci are Gram-positive bacteria from the family Staphylococcaceae and phylum

Firmicutes. They form a common part of skin microbiota, mucous membrane and digestive tract

of animals [1]. Based on the plasma coagulation ability, staphylococci are divided into

coagulase-negative and coagulase-positive [2]. Some strains of the family Staphylococcaceae

can possess genes for virulence factors [3]. Staphylococci are recognized as the most frequent

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European Journal of Applied Sciences (EJAS) Vol. 12, Issue 1, February-2024

causes of biofilm associated infections [4]. Bacteria that grow in the form of a biofilm are

characterized by increased resistance to host defense responses as well as natural resistance

to antibiotic activity [5]. The development of biofilm is influenced by many environmental

conditions such as pH, ambient temperature, presence of nutrients and oxygen concentration

[6, 7]. Planktonic cells are capable of adhering to surfaces using adhesins, i.e., surface proteins.

For biofilm formation is also important the presence of a gradient between the surface of

bacteria and the material that allows better attachment of bacteria [8]. The formation of biofilm

is related to antibiotic resistance, and it can disrupt even the trade with animals and animal

products because it affects the health and living conditions of animals, and thus their

productivity [9]. In our study, the most impact was focused on biofilm formation ability as one

of virulence factors in coagulase-negative staphylococci (CoNS). Staphylococci are

characterized by commensal incidence in animals. Nowadays, 66 species have been validated

(http://www.bacterio.ne) [10]. Under certain conditions, some representatives of the genus

Staphylococcus are diseases-causing agents in animals; e.g., skin dermatitis. The aim of this

study was to test biofilm-forming ability in coagulase-negative staphylococci isolated from

faeces of different animals; biofilm can make them potential agents threatening animal health.

Up to now, the target of CoNS from so many different animals has not been tested for biofilm- forming ability yet. Moreover, their antibiotic phenotype was tested.

MATERIAL AND METHODS

A total, 100 faecal staphylococcal strains of different species (Staphylococcus capitis, S. caprae,

S. cohnii, S. epidermidis, S. equorum, S. haemolyticus, S. hominis, S. lentus, S. pasteuri, S.

saprophyticus, S. sciuri, S. succinus, S. vitulinus, S. warneri, S. xylosus) were tested. They were

isolated from faeces of 407 different animals, including hens (8, Gallus gallus domesticus),

broiler rabbits (155, Oryctolagus cuniculus domesticus-breed M91, Hyla or Hyplus lines),

pheasants (60, Phasianus colchicus), ostriches (140, Struthio camelus), horses (32,

Cabballus/Equs, Slovak breed Norik from Muráň, Hucul breed, Polish warm-blooded, British

blood-horse), and deer (12, Capreolus capreolus). Faeces were sampled from private breeding

husbandries, in aviaries, at farms and during application experiments. Coagulase-negative

staphylococci (CoNS) from horses, pheasants, ostriches and deers were isolated as previously

reported by Lauková and Kandričáková [11], Lauková et al. [12], Kandričáková et al. [13].

(2016) and Bino et al. [14]. The strains were stored with the MicrobankTM system (Pro-Lab

Diagnostic, USA).

Antibiotic Phenotype Testing

Antimicrobial profile was tested using the disc diffusion method on Mueller-Hinton agar

(Oxoid) according to CLSI [15]. The antibiotic discs (13, Oxoid) from different antibiotic groups

were used. Penicillin (10 μg) and ampicillin (10 μg) belong to β-lactams. Aminoglycosides group

was represented by gentamicin (120 μg). Macrolid antibiotic erythromycin (15 μg) was

involved in testing. A broad-spectrum antibiotic such as chloramphenicol (30 μg) and

tetracycline (30 μg) were also involved in testing. Fluroquinolon ciprofloxacin (5 μg) was tested

as well as oxazolidinon antibiotic linezolid (30 μg). Also, quinupristin-dalfopristin (15 μg) was

used. Peptidoglycan teicoplanin (30 μg) was involved in testing and ansamycin antibiotic

rifampicin (30 μg) as well. Vancomycin is glycopeptide (30 μg). Moreover, trimethoprim (5 μg)

was involved in the test. Agar plates with broth cultures (100 μl) of tested strains and applied

antibiotic discs were incubated at 37°C overnight. After incubation, the inhibitory zones were

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Bino, E., Kandričáková, A., & Lauková, A. (2024). Biofilm Formation in Coagulase-Negative Staphylococci from Various Animals. European Journal of

Applied Sciences, Vol - 12(1). 317-324.

URL: http://dx.doi.org/10.14738/aivp.121.16462

measured and expressed in mm. They were interpreted as resistant, or susceptible based on

the Clinical and Laboratory Standards Institute breakpoint table [15]. Staphylococcus aureus

CCM 44 was used as the control strain.

Biofilm -Forming Evaluation Using Different Methods

Biofilm formation in identified staphylococcal strains was tested by three different methods;

two qualitative phenotypic methods were used and one quantitative method. First qualitative

method is based on biofilm formation testing on Congo red agar [16]. The components of

cultivation medium formed Brain-heart infusion (Difco, Michigan, USA, 37 g/l) enriched with

sucrose (36 g/l), pure agar (30 g/l) and Congo red dye (0.8 g/l, Merck, Germany). Staphylococci

were inoculated on Congo red agar and incubated at 37° for 24 hours. Biofilm formation was

demonstrated by black colonies with a dry crystalline consistency. Non-slime producers usually

remained pink. The color was also checked after 48 and 72 hours.

The second qualitative method was the modified tube method [17]. Brain-heart infusion (Difco)

in glass tubes was inoculated with one colony of overnight culture of tested strain on blood

agar. After incubation (37°C, 24 hours), the tubes were removed, washed in phosphate buffer

(pH 7.4) and dried. After drying, each tube was dyed with 0.1 % solution of crystal violet.

Subsequently, each tube was gently rotated to ensure if staining was on the inner surface of the

tube, and then the tube contents were gently tumbled. The tubes were placed upside down in a

rack. Biofilm-formation was indicated by the presence of an adherent layer of stained material

on the inner surface of the tubes, and then evaluated as 0, low-grade positive (1) and high-grade

positive (2) slime (biofilm) formation.

The quantitative method was performed in microtiter plates as previously described and

evaluated by Chaieb et al. [8] and Slížová et al. [18]. One colony of the tested strain grown

overnight at 37°C in Brain-Heart Infusion (BHI, Difco) was transferred into 5 ml of Ringer

solution (pH 7.0, 0.75% w/v) to reach the McFarland standard 1 suspension that corresponded

to 1 x 108 cfu/ml. A volume of 100 μl was then transferred into 10 ml of BHI. That standardized

culture (200 μl) was inoculated in a well on a polystyrene microtiter plate (Greiner ELISA 12

Well Strips, 350 μl, flat bottom, Frickenhausen GmbH, Germany) and incubated at 37°C for 24

h. The biofilm formed in the well of the microtiter plate well was washed twice with 200 μl of

deionized water and dried at 25°C for 30 min in an inverted position. The remaining attached

bacteria were stained for 30 min at 25°C with 200 μl of 0.1 % (m/v) crystal violet in deionized

water. The dye solution was aspirated away and the wells were washed twice with 200 μl of

deionized water. After water removal and drying for 30 min at 25°C, the dye bound to the

adherent biofilm was extracted with 200 μl of 95% ethanol and stirred. A 150 μl aliquot was

transferred from each well and placed in a new microtiter plate for absorbance (A)

determination at 570 nm using a Synergy TM4 Multi Mode Microplate reader (Biotek USA). In

each method, the positive control strain was Streptococcus equi subsp. zooepidemicus CCM 7316

(kindly provided by Dr. Eva Styková, University of Veterinary Medicine and Pharmacy in Košice,

Slovakia). Biofilm formation was then classified according to Chaieb et al. [8] and Slížová et al.

[18] as highly-grade positive (A570 ≥1) and/or low-grade positive (0.1 ≤ A570 < 0.1). Using of

three methods allows better evaluation of biofilm formation in staphylococci. We don’t follow

their comparison.