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Article

Gram-Negative Microbiota Derived from Trout Fished in Slovakian Water Sources and Their Relationship to Postbiotics

by
Andrea Lauková
1,*,
Anna Kandričáková
1,2,
Jana Ščerbová
1,
Monika Pogány Simonová
1 and
Rudolf Žitňan
3
1
Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6, 040 01 Košice, Slovakia
2
Parasitological Institute, The Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovakia
3
National Agricultural and Food Centre, Research Institute for Animal Production, Hlohovecká 2, 951 41 Nitra-Lužianky, Slovakia
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(7), 644; https://doi.org/10.3390/pathogens14070644
Submission received: 13 June 2025 / Revised: 17 June 2025 / Accepted: 24 June 2025 / Published: 28 June 2025
(This article belongs to the Section Bacterial Pathogens)
Versions Notes

Abstract

Regarding the trout microbiota, most information is focused on lactic acid bacteria, which can show beneficial properties. However, in trout farming, mostly pathogenic Gram-positive species were reported, such as Staphylococcus aureus, Listeria monocytogenes, and/or Clostridium spp. In this study, free-living trout were analyzed for Gram-negative microbiota that can cause loss as disease-stimulating agents. Bacteriocin postbiotics should be one of the approaches used to eliminate these agents. In total, 21 strains of different species isolated from the intestinal tract of 50 trout in Slovakia (Salmo trutta and Salmo gairdnerii) were taxonomically allotted into 13 species and 9 genera. This method showed variability in microbiota identified using MALDI-TOF mass spectrometry with the following species: Acinetobacter calcoaceticus, Citrobacter gillenii, Citrobacter freundii, Escherichia coli, Hafnia alvei, Kluyvera cryocrescens, K. intermedia, Leclercia adecarboxylata, Raoultella ornithinolytica, Pseudomonas fragi, Ps. putida, Ps. lundensis, Ps. teatrolens, and Serratia fonticola. Most strains were susceptible to the antibiotics used, reaching inhibitory zones up to 29 mm. On the other hand, 3 out of 21 strains (14%) were susceptible to nine enterocins- postbiotics (Hafnia alvei Hal281, Pseudomonas putida Pp391, and Ps. fragi Pf 284), with inhibitory activity in the range of 100–6400 AU/mL.

1. Introduction

Freshwater hosts a substantial source of animal protein in the human diet. Nowadays, the growing importance of aquaculture in the production of animal-originating proteins has been noted [1]. Trout can contribute to this source. It belongs to the numerous species of carnivorous freshwater ray-finned fishes. As is generally known, this fish species is taxonomically involved in the genus Salmo and the family Salmonidae. Trout as a species is classified as an oily fish and has been an important food fish for humans [2]. Oily fish refer to fish species with oil (fats) in soft tissues and in the coelomic cavity around the gut. Fillets of these fishes may contain up to 30% oil, although this figure varies both within and between species [2]. Oily fish can be contrasted with whitefish (e.g., cod), which contain oil only in the liver and in a much smaller overall quantity than oily fish. Oily fish meat is a good source of important fat-soluble vitamins such as vitamin A and D, and it is rich in omega-3 fatty acid (white fish also contain these nutrients but at a much lower concentration). For this reason, the consumption of oily fish rather than white fish can be more beneficial to humans, particularly in relation to heart diseases such as stroke and ischemic heart disease [2]. The omega-3 fatty acids in oily fish may help improve inflammatory conditions such as arthritis [2]. However, oily fish are known to carry higher levels of contaminants than white fish [2].
Nowadays, there is also increased concern about trout farming. The main challenges of small-scale farmers are seed availability and maintenance, growth, and reproduction functions. Fish use mostly dietary proteins continuously, and the availability of quality feeds is the main challenge faced by the farmers. However, there exist many factors from catch to processing that influence the natural condition of the fish when it is captured and handled [3]. The microbial aspect is among those factors.
The intestinal microbiota of fish (trout included) is often reported to be highly variable [4]. Of course, it depends on several factors, including environmental and temperature conditions. The majority of research is focused on lactic acid bacteria (LAB), which can show beneficial properties, including bacteriocinogenic characteristics [5]. The interest in the usage of beneficial LAB is associated mostly with fish farming in the form of trout production, which accounts for about 70% in Slovakia [5,6,7]. Regarding pathogens, mostly Gram-positive species have been mentioned, such as Staphylococcus aureus, Listeria monocytogenes, and/or Clostridium spp. [7]. Although there are existing microbial analyses of trout based on different identification systems and levels, there is still a gap in knowledge. However, in our case, free-living trout were analyzed on Gram-negative microbiota, because less interest is focused on mapping non-essential Gram-negative microbiota. Through farming, but also in free-living fish, massive losses can be caused by diseases due to pathogenic (mostly Gram-negative) bacteria [4,8,9]. In addition, the aquatic environment plays a preponderant role in the composition of the microbial community constituting the fish gut microbiota [1,9,10,11]. For example, Kim et al. [10] reported a high percentage abundance of the phyla Proteobacteria and Actinobacteria in rainbow trout. Three morphological types of Gram-negative, oxidase-positive bacteria were identified in fish by Grigoryan et al. [3].
Semwal et al. [9] also reported the representatives of Gammaproteobacteria as disease agents in aquaculture. Among them, the species Aeromonas hydrophila has been frequently detected [9], which was also referred to in our previous study [12]. This species is usually considered a secondary pathogen that infects fish that have already been infected with another pathogen, and/or as an opportunistic invader infecting fish under stress conditions. It is also an efficient biomarker of a stressed or polluted aquatic environment [9].
One strategy/approach on how to prevent/reduce contaminants in fish can be represented by probiotic/beneficial bacteria and/or substances produced by them—bacteriocins [5,13,14,15,16,17,18]. Nowadays, bacteriocins are part of the group of postbiotics. Postbiotics are defined as preparations of inanimate microbiota and/or their components, conferring health to the host [16]. They have been mostly explored as therapeutics in veterinary medicine for addressing bovine mastitis [19]. Another explored application of bacteriocins is as a preventive or growth promoter in broiler production for controlling pathogens or non-beneficial microbiota [19,20,21,22,23]. In this case, especially postbiotics, enterocins characterized in our laboratory were applied, e.g., enterocin (Ent) M, a thermo-stable small peptide produced by the environmental strain E. faecium AL41 (CCM8558), which was seen to achieve beneficial effects in rabbits [19,20,21,22,23]. Increased phagocytic activity in blood samples and a decrease in the fecal coliforms, S. aureus, and enterococci were noted after 21 days of the application of EntM in broiler rabbits. Moreover, higher daily weight gain was noted with EntM application, although no influence on feed conversion was seen [23].
Keeping in mind these benefits, and based on our previous in vitro and in vivo studies with postbiotics, one aim of this study was to show variability regarding the Gram-negative representatives derived from free-living trout originating from Slovakian water sources and to show if postbiotic (enterocin) treatment can inhibit them to support a sustainable health strategy in aquaculture, because some Gram-negative species can cause disease due to being a stimulating agent. Therefore, the use of postbiotics to inhibit them seems to be an alternative innovative approach following the One Health concept.

2. Materials and Methods

2.1. Sampling and Species Strain Identification

Fifty (n = 50) healthy trout (Salmo trutta and Salmo gairdnerii) were sampled from different sides of the regular pond Bukovec near Košice in eastern Slovakia and/or the river Čierny Váh in Central Slovakia in April of the years 2012–2015. Trout were supplied by individual fisherman who had obtained the appropriate permission for fishing to follow scientific research and exploratory purposes. Trout were fished based on the special fishing permit for scientific research and exploratory purposes (No. 42/2012, No. 21/2013, No. 4/2014, and No. 1/2015 provided by the Ministry of Environment of the Slovak Republic). The intestinal samples of trout were collected at the place of the trout fishing formerly described by Lauková et al. [12]. Samples were taken aseptically from the fish and placed into sterile Petri dishes, stored at 4 °C for approximately 4 h, and transported to the laboratory. They were treated using the standard microbial dilution method specified by the ISO (International Organization for Standardization). The samples were stirred in Ringer solution (1:9, pH 7.0, Merck, Darmstadt, Germany). The appropriate dilutions were plated onto different agars, such as CLED agar (Merck, Darmstadt, Germany), Mac Conkey agar for enterobacteriae (Difco, Sparks, MD, USA), and/or Cetrimide agar (Difco, USA). The agar plates were cultivated at 28–37 °C for 24–48 h according to the type of selected media. The grown colonies from the highest dilution were randomly picked up, their purity was controlled by plating on Trypticase soy agar with blood (Difco, USA), and their morphology was assessed. Strain identification was performed with the use of MALDI-TOF mass spectrometry (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry). This is an accurate method used in laboratory practices. This method has a very simple approach designed for protein detection and reliable technology for the precise identification of cultured microorganisms [12]. For MALDI-TOF mass spectrometry, a solitary colony of each isolate from Trypticase soy agar (supplemented with 3% defibrinated sheep blood, Difco, Sparks, MD, USA) was mixed with a matrix (α-cyano-hydroxycinnamic acid and trifluoroacetic acid). The suspension was spotted onto a MALDI plate and ionized with a nitrogen laser (wavelength of 337 mm; frequency of 20 Hz). This identification method is based on protein “fingerprints” (Bruker Daltonics, Biotyper 2.2. [12,24,25]), performed using a Microflex MALDI-TOF mass spectrometer, as previously described by Lauková et al. [12]. The results were evaluated according to the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA) identification database. Taxonomic classification was assessed on the basis of highly probable species identification (a value score of 2.300–3.000), secure probable species identification/probable species identification (2.000–2.299), and on the basis of highly probable genus identification (1.700–1.999). Positive controls were those involved in the identification system. Identical colonies evaluated using the MALDI-TOF MS score value were excluded. Finally, 21 strains were used for the next testing. They were stored for analyses conducted with the Microbank system (Pro-Lab Diagnostic, Richmond, BC, Canada).

2.2. Antibiotic Phenotype of the Identified Species Strains

The commercial antibiotic discs supplied by Oxoid Ltd. (Basingstoke, UK) were used in the disc diffusion test. They were decided according to CLSI [26]. The following antibiotic discs were tested: clindamycin (2 µg), ampicillin (10 µg), penicillin G (10 IU), erythromycin, azithromycin (15 µg), chloramphenicol, tetracycline, amikacin (30 µg), azlocillin, mezlocillin, ticarcillin (75 µg), trimethoprim (85 µg), carbenicillin, piperacillin 100 µg), and gentamicin (120 µg). Broth cultures (in Trypticase soy broth, Difco) of tested strains (100 µL) were spread onto Mueller–Hinton agar (Bio-Rad, Bratislava, Slovakia) supplemented with 3% of sheep blood. The antibiotic discs were applied to the agar surface. The plates were incubated at 37 °C for 18 h. The inhibitory zones were evaluated and expressed in mm. They were reported according to the guidelines of the Clinical and Laboratory Standards Institute [15]. E. coli ATCC 25922 was the control strain.

2.3. Testing Gram-Negative Species Strains for Their Susceptibility to Postbiotics

The precipitates (partially purified substances) from our nine producing strains were used. Enterocin–dipeptide (Ent) A/P was produced by the environmental strain EK13 (CCM 7419 [27]). Ent A/P is a thermo-stable dipeptide with a molecular mass of 4830 Da. Its optimum production is associated with the pH range of 5.0–6.5 at 30 °C and 37 °C. The most active substance is produced in the logarithmic growth phase of the producer strain. It has a broad inhibitory spectrum. EntM, similarly to EntA/P, is produced by the environmental strain E. faecium AL41 = CCM8558. It is a thermo-stable, small peptide (molecular mass 4628 Da), with a broad antimicrobial spectrum [18]. Ent412, produced by the horse-derived strain E. faecium EF 412, indicates a broad inhibitory spectrum [28]. Ent4231, produced by the ruminal strain E. faecium CCM4231 [29], has been shown to have a broad antimicrobial spectrum [29]. It is susceptible to pronase and thermo-resistant. Ent55, produced by the poultry strain E. faecium EF [30] is a thermo-stable bacteriocin with the highest production in the late logarithmic growth phase, with optimal production at pH levels ranging from 7.0 to 9.0. Its inhibitory activity is mostly against Gram-positive microbiota. Ent2019 is produced by the rabbit-derived strain E. faecium 2019-CCM7420 [23], and Ent 9296 is from the silage strain E. faecium EF9296 [31]. EntEM41 is produced by the strain E. faecium EM41 from ostrich [22], and Durancin ED26E/7 is produced by the milk lump cheese-derived strain E. durans ED26E/7 [32,33]. Enterocins were prepared according to the previously reported protocols. They are class II enterocins. Testing was performed using the agar spot method according to De Vuyst et al. [34]. In brief, BHagar (Difco) was basic agar. Overnight, a broth culture of the EA5 strain (A600 up to 1.0) (200 µL) was applied into 0.7% BHagar, and the plate was overlaid. The appropriate dilutions of enterocins (in phosphate buffer, pH 6.5, 10 µL) were spotted on the agar plate surface. The plates were stored for 10 min in the fridge to diffuse the enterocin. Then, they were incubated at 37 °C overnight. The inhibitory activity was expressed in arbitrary units per mL (AU/mL). This means a two-fold dilution (in phosphate buffer pH 5.5) of the precipitate, which inhibited the growth of the indicator strain. The initial activity of the precipitate was measured against the principal indicator E. avium EA5—our strain (EntM, Ent2019, Ent 9296—25,600 AU/mL, EntA/P, Ent 55—51,200 AU/mL, Ent 412—204,800 AU/mL, ED26E/7 Ent 4231—6400 AU/mL, and EM41—800 AU/mL).

3. Results

3.1. Identified Species Strains

Broad species variability was found among trout-derived Gram-negative strains. A total of 21 strains were taxonomically allotted to 13 species and 9 genera. The following species were detected (Table 1): Acinetobacter calcoaceticus, Citrobacter gillenii, Citrobacter freundii, Escherichia coli, Hafnia alvei, Kluyvera cryocrescens, K. intermedia, Leclercia adecarboxylata, Raoultella ornithinolytica, Pseudomonas fragi, Ps. putida, Ps. lundensis, Ps. teatrolens, and Serratia fonticola. Six variable species strains (6) were identified, reaching a score value in the range of 2.300–3.000 (Table 1), meaning highly probable species identification. The species Hafnia alvei Hal401 (2.487), Kluyvera intermedia Ki382 (2.425), K. intermedia Ki 371 (2.326), Raoultella ornithinolytica Ro381 (2.468), Pseudomonas putida Pp391 (2.300), and E. coli EC361 (2.435) were also identified. Most of the strains were identified with a score in the range of 2.000–2.299 (Table 1), involving 11 different species identified based on secure probable species identification/probable species identification. A score of 1.700–1.999 was found for four strains, Ps. fragii Pf284 and Pf302, Ps. teatrolens Pt285, and Leclercia adecarboxylata LA381 (Table 1). The strains identified from trout sample No. 36 included four different species: Acinetobacter calcoaceticus AC362, Escherichia coli EC361, Ps. lundensis PL361, and Serratia fonticola SF361. The species K. intermedia Ki371, Citrobacter gillenii CG372, and H. alvei Hal 371 were detected in trout sample No. 37. Six species, such as C. gillenii CG382, H. alvei Hal382, K. cryocrescens Kc383, K. intermedia Ki382, Leclercia adecarboxylata LA381, and R. ornithinolytica Ro381, were derived from trout sample No. 38. The strains of the species Ps. teatrolens Pt285, Ps. fragi Pf284, and H. alvei Hal281 were also isolated from the same trout. In trout No. 40, H. alvei Hal401 and C. freundii CF402 were determined. In trout No. 30, Ps. fragi Pf302 was found. H. alvei Hal331 was detected in trout No. 33, and Ps. putida Pp391 was found in a sample of trout No. 39. The most prevalent Gram-negative species among trout were Hafnia alvei and different species of the genus Pseudomonas (Table 1). Trout No. 28 and 30 were caught in Čierny Váh; the others, No. 33–40 trout, were caught in the pond in Bukovec.

3.2. Susceptibility to Antibiotics and Postbiotics

The strains were treated with 15 antibiotics according to CLSI [26]. Twenty-one tested strains (21) were susceptible to mezlocillin, with an inhibitory zone size in the range of 15–23 mm. They were also susceptible to tetracycline (17–20 mm), chloramphenicol (22–27 mm), piperacillin (17–29 mm), gentamicin (16–26 mm), amikacin (10–22 mm), azlocillin (12–22 mm), and azithromycin (10–20 mm). In contrast, 21 strains were resistant to penicillin. Almost all strains (except Hal281) were resistant to erythromycin and clindamycin (Table 2). Altogether, Gram-negative bacteria tested were mostly susceptible to the antibiotic used; all were at least susceptible to 8 out of 15 antibiotics, and most were susceptible to 13 out of 15 antibiotics. Among the tested strains, three (3) were susceptible to 12 antibiotics (resistant to penicillin). Hal281 was the most susceptible (to 13 antibiotics out of 15 used). Nine strains were susceptible to 10 antibiotics, and seven strains were susceptible to 9 antibiotics. Even LA381 (Table 2) was susceptible to 8 antibiotics out of 15 tested. Only two strains were resistant to ticarcillin (LA381, Hal331).
Regarding postbiotic treatment, 3 out of 21 strains were susceptible to enterocins-postbiotics. The strains included Hafnia alvei Hal281, Pseudomonas putida Pp391, and Ps. fragii Pf 284 (Table 3). Pseudomonads were inhibited, with Ents reaching inhibitory activity of 100 AU/mL. However, Hafnia alvei Hal281 was the most susceptible. In the case of EntA/P, Ent9296, Ent412, Ent2019, and Durancin-like substance ED26E/7, inhibitory activity reached 6400 AU/mL. The other Ents inhibited Hal281, with inhibitory activity of 3200 AU/mL. This means that only 3 out of 21 different strain species (14%) were susceptible to nine of the postbiotics used. Eighty-six (86%) Gram-negative species strains were not inhibited by the Ents (postbiotics) used.

4. Discussion

The strain Acinetobacter calcoaceticus AC362 is part of the phylum Pseudomonata, class Gammaproteobacteria, order Pseudomonales, family Moraxellacae, and genus Acinetobacter [35]. To the same phylum and class, the species strain Kluyvera cryocrescens Kc383 and the strains K. intermedia, Ki382, and Ki372 were also allotted. They are, however, from the order Enterobacterales, family Enterobacteriacae, and genus Kluyvera [36]. The species Citrobacter gillenii and C. freundii (CG372, CG382, and CF402) also belong to the same family (Enterobacteriacae) [37]. The species E. coli EC361 also belongs to the same order and family, but to the genus Escherichia [38]. Raoultella ornithinolytica Ro381 belongs to the order Enterobacteriales, family Enterobacteriacae, and class Gammaproteobacteria; however, they belong to the phylum Pseudomonata [39]. Hafnia alvei is the species belonging to the phylum Pseudomonata, class Gammaproteobacteria, order Enterobacterales, family Hafniacae, and genus Hafnia [40]. The species Leclercia is a re-classified type of bacterium [41]. This bacterium was known as Escherichia adecarboxylata, and it is known as a human pathogen, which, however, is mostly susceptible to antibiotics [42]. Their taxonomy is also included in the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriacae, and genus Leclercia. The same category is related to the species Serratia fonticola SF361. However, Serratia is allotted to the family Yersiniacae and the genus Serratia [43]. Pseudomonads belong to the same phylum and class, although they belong to the order Pseudomonales, family Pseudomonacae, and genus Pseudomonas [44]. In summary, five out of nine detected bacterial genera were from the phylum Proteobacteria (Citrobacter, Pseudomonas, Serratia, Leclercia, and Escherichia), and four genera belonged to the phylum Pseudomonata (Kluyvera, Raoultella, Acinetobacter, and Hafnia). However, all strains of detected species belong to the class Gammaproteobacteria. Seven genera (Citrobacter, Escherichia, Kluyvera, Raoultella, Serratia, Hafnia, and Leclercia) belong to the order Enterobacterales, and two genera (Acinetobacter and Pseudomonas) belong to the order Pseudomonales. The species of detected strains belong to five (5) families: Enterobacteriacae, with five genera (Citrobacter, Escherichia, Kluyvera, Leclercia, and Raoultella); Moraxellacae (Acinetobacter); Pseudomonacae (Pseudomonas); Yersiniacae (Serratia); and Hafniacae (Hafnia). In addition, detected bacteria belong to two phyla, Proteobacteria and Pseudomonata. Kluyvera spp. often occur in water and sewage and can be dangerous for immune-compromised patients [36,44,45]. However, for example, studying Hafnia alvei has been recommended in terms of two aspects: beneficial and non-requested. It can have importance because the production of a protein called caseinolytic protease B, which has been shown to be a mimetic of the hormone α-MSH, which is implicated in satiety [46]. From pathogenicity, Hafnia is considered a secondary pathogen via the nosocomial route. As has already been mentioned, L. adecarboxylata acts as a human pathogen; however, it is susceptible to antibiotics as effective therapy [46,47]. Serratia has been identified as an opportunistic pathogen. This means it can cause infection in individuals with weakened immunity or underlying medical conditions. This species is able to develop resistance to multiple antibiotics [48]. However, in our study, this strain, SF361, was susceptible to 9 out of 15 antibiotics. Johns et al. [38] reported a high prevalence of resistant E. coli in horses, and it is necessary to minimize their potential to be spread. Following this aspect, the use of postbiotics is promising, although in this study, only one strain E. coli was tested and it was postbiotic-resistant. However, in vivo, coliforms were reduced in animal experiments related to postbiotic–bacteriocin application [15]. Similarly, Citrobacter spp. is an opportunistic pathogen [48]. Raoultella spp. has been reported to be generally sensitive to treatment with carbapenems and β-lactamases [48]. Pseudomonas spp. is a bacterium widespread in various environments, and it has been shown that MALDI-TOF MS analysis is a reliable methodology for its identification, with up to 100% of isolates correctly identified [24]. Because there are more than 100 validated Pseudomonas species, there is also a problem with antibiotic resistance, and here, a novel approach seems to be postbiotics.
MALDI-TOF mass spectrometry has been indicated as a revolutionized procedure for bacterial clinical identification, as reported by Fedorko et al. [47]. In our study, broad variability in Gram-negative microbiota was detected. We did not include aeromonads; however, their species variability was presented in trout isolated from Slovakian water sources using MALDI-TOF MS identification in our previous study [12]. There were 9 different species determined among 25 isolated strains. Moreover, similarly to here, only two strains (aeromonads) were inhibited by the use of enterocins-postbiotics; the strains Aeromonas bestiarum R41/1 and R47/3 were inhibited with inhibitory activity of 100 AU/mL. Novotný et al. [8] described Gram-positive and Gram-negative pathogens from fishes, as well as Gram-negative species such as Aeromonas spp., Campylobacter spp., Vibrio spp., and Salmonella spp. They are also frequent agents that cause human infections. Regarding antibiotic susceptibility, the aeromonads formerly mentioned were similar to Gram-negative bacteria in terms of susceptibility [8]. However, there was noted resistance to ticarcillin of aeromonads, but in our study, strains were mostly susceptible to ticarcillin. In free-living trout, susceptibility to antibiotics should be obligatory, unlike farmed trout; farmed trout are commonly exposed to the use of antibiotics to prevent a loss caused by diseases [8,48]. Semwal et al. [9] reported the effectiveness of medicinal herbs against aeromonads in aquaculture. Phytotherapy-based techniques appear to be inexpensive and environmentally friendly, and bacteriocins (postbiotics) are. The plant extract from water hyacinth appeared to be effective against Streptococcus iniae in trout [9]. In general, there is an increasing interest in controlling antibiotic use. An alternative method to maintain a healthy microbial environment could be the use of postbiotics. However, more effectively, one could select postbiotics with a broad antimicrobial spectrum to eliminate Gram-negative bacteria as potential pathogens. Even Hafnia alvei, the strain HA4597, was reported to have an effect on cholesterol levels and induced a change in blood sugar levels, and it was indicated as a “precision probiotic” [49] with the potential to produce bacteriocin. Moreover, the antimicrobial effect of postbiotics has already been declared in vitro and in vivo [13,15]. Even the in vitro growth of fecal pseudomonads was inhibited using EntA/P, EntM, Ent55, and Ent2019 [50,51]. As reported, up to 9 out of 19 strains were inhibited with an inhibitory activity of 100 AU/mL. In vivo, the effect of postbiotic/enterocins was not only noted in broiler animals but also, e.g., in horses. After EntM administration for 21 days, an increased tendency of phagocytic activity in blood and a reduction in coliforms, campylobacters, and clostridiae was noted [15]. Selecting new bacteriocins produced by some of those species probably enables more effective inhibition of individual Gram-negative bacteria. Moreover, more effective inhibition could probably be achieved by using autochthonous strains that produce novel antimicrobial peptides, as declared, e.g., by Fan et al. [52]. Moreover, more intensive studies have been conducted to clarify the mode of action of postbiotics used in various applications [53]. For each case, our point of view represents an innovative approach.

5. Conclusions

Twenty-one (21) species strains isolated from the intestinal tract of trout in Slovakia were taxonomically allotted to 13 species and 9 genera, including the following species: Acinetobacter calcoaceticus, Citrobacter gillenii, Citrobacter freundii, Escherichia coli, Hafnia alvei, Kluyvera cryocrescens, K. intermedia, Leclercia adecarboxylata, Raoultella ornithinolytica, Pseudomonas fragi, Ps. putida, Ps. lundensis, Ps. teatrolens, and Serratia fonticola. The strains were mostly susceptible to antibiotics, and 14% of strains tested were susceptible to nine enterocins-postbiotics, with inhibitory activity up to 6400 AU/mL. An innovative approach to studying postbiotic use in aquaculture has been proposed. However, additional application experiments are required.

Author Contributions

Conceptualization, A.L.; methodology, A.L., A.K., J.Š. and M.P.S.; validation, A.L.; investigation, A.L.; data curation, A.L.; writing—original draft preparation, A.L.; writing—review and editing, A.L.; supervision, A.L.; project administration, A.L. and M.P.S.; resources, R.Ž. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the VEGA projects 2/0006/2017 and partially by 2/0009/25.

Institutional Review Board Statement

Ethical review and approval were waived for this study because the material was received from our colleague. However, trout were fished based on the special fishing permit for scientific research and exploratory purposes, No. 42/2012, No. 21/2013, No. 4/2014, and No. 1/2015 for Rudolf Žitňan, provided by the Ministry of Environment of the Slovak Republic.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be provided by the corresponding author upon request.

Acknowledgments

I would like to thank Margita Bodnárová for her laboratory skills.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Gram-negative species originated from trout identified using MALDI-TOF mass spectrometry.
Table 1. Gram-negative species originated from trout identified using MALDI-TOF mass spectrometry.
SpeciesSampleScoreIndication
A. calcoaceticusR36/22.225AC362
C. gilleniiR37/22.319CG372
C. gilleniiR38/22.330CG382
C. freundiiR40/22.082CF402
E. coliR36/12.435EC361
H. alveiR33/12.108Hal331
H. alveiR37/32.126Hal373
H. alveiR38/22.273Hal382
H. alveiR40/12.487Hal401
H. alveiR28/12.051Hal281
K. intermediaR38/22.425Ki382
K. intermediaR37/12.326Ki371
L. adecarboxylataR38/11.788LA381
R. ornithinolyticaR38/12.468Ro381
Ps. teatrolensR28/51.856Pt285
Ps. putidaR39/12.300Pp391
Ps.fragiR28/41.887Pf284
Ps. fragiR30/21.947Pf302
Ps. lundensisR36/12.225PL361
S. fonticolaR36/12.256SF361
A. calcoaceticus, Acinetobacter calcoaceticus; C. gillenii, Citrobacter gillenii; C. freundii, Citrobacter freundii; E. coli, Escherichia coli; H. alvei, Hafnia alvei; K. intermedia, Kluyvera intermedia; K. cryocresens, Kluyvera cryocrescens; L. adecarboxylata, Leclercia adecarboxylata; R. ornithinolythica, Raoultella ornithinolytica; Ps. teatrolens, Pseudomonas teatrolens, Ps. putida, Pseudomonas putida; Ps. fragi, Pseudomonas fragi, Ps. lundensis, Pseudomonas lundensis, S. fonticola, and Serratia fonticola. Highly probable species identification (value score of 2.300–3.000), secure probable species identification/probable species identification (2.000–2.299), and highly probable genus identification (1.700–1.999).
Table 2. Antibiotic profile (phenotype) of Gram-negative trout-derived strains.
Table 2. Antibiotic profile (phenotype) of Gram-negative trout-derived strains.
StrainCarbenicillin (100 µg)Erythromycin (15 µg)Ampicillin (10 µg)Trimetroprim (85 µg)Ticarcillin (75 µg)Clindamycin (2 µg)
AC362RRRR+12R
CG372RR+10R+11R
CG382RR+12R+10R
CF402RR+10R+12R
EC361+15R+12R+15R
Hal281+14+15+21R+20+19
Hal331RR+11RRR
Hal373RRRR+12R
Hal382RR+10R+12R
Hal401RRRR+11R
Kc383RR+10R+10R
Ki382RR+10R+13R
Ki371+20R+20+16+26R
LA381RRRRRR
Ro381RR+11R+13R
Pt285+14R+15+12+19R
Pp391RRRR+11R
Pf284RR+11R+12R
Pf302RR+16R+11R
PL361+15R+17+11+20R
SF361RRRR+10R
The strains were susceptible to mezlocillin (MEZ—75 µg), tetracycline (TE—30 µg), chloramphenicol (C—30 µg), piperacillin (100 µg), gentamicin (GN—120 µg), amikacin (AK—30 µg), azlocillin, mezlocillin (AZ, MEZ—75 µg), and azithromycin (AZM—15 µg). The strains were resistant to penicillin (P-10 IU); R—resistant; +—susceptible to inhibitory zone size in mm; AC36—Acinetobacter calcoaceticus AC362, CG—Citrobacter gillenii CG372, CG382, CF—C. freundii CF402; EC—Escherichia coli EC361; Hal—Hafnia alvei; Kc—Kluyvera cryocrescens Kc383, Ki—Kluyvera intermedia Ki382, Ki381; LA—Leclercia adecarboxylata LA381; Ro381—Raoultella ornithinolytica; Pt—Pseudomonas teatrolens Pt285; Pp391—Ps. putida, Pf 284, Pf302—Ps. fragi; PL361—Ps. lundensis, SF361—Serratia fonticola.
Table 3. Enterocin postbiotics used for treatment of Gram-negative strains and inhibition of 3 strains (arbitrary units per milliliter, AU/mL).
Table 3. Enterocin postbiotics used for treatment of Gram-negative strains and inhibition of 3 strains (arbitrary units per milliliter, AU/mL).
StrainsEntA/PEntMEnt4231Ent55Ent9296Ent412Ent2019EntEM41DurED26E/7
Hal28164003200200320064006400640032006400
Pp391100100100100100100100100100
Pf284100100100100100100100100100
Hal281, Hafnia alvei 281, Pp, Pseudomonas putida 391, Pf, Ps. fragi 284; EntA/P, enterocin-dipeptide produced by the environmental strain EK13-CCM7419 [27]; EntM, produced by the environmental strain E. faecium AL41-CCM8558 [18]; Ent412, produced by the horse-derived strain E. faecium EF 412 [28]; Ent4231, produced by the ruminal strain E. faecium CCM 4231 [29]; Ent55, produced by the poultry strain E. faecium EF55 [30]; Ent2019, produced by the rabbit-derived strain E. faecium 2019 -CCM 7420) [21]; Ent9296, produced by silage strain E. faecium EF9296 [31]; EntEM41, produced by ostrich strain E. faecium EM41 [22]; and Durancin ED26E/7, produced by the milk lump cheese-derived strain E. durans ED26E/7 [33]. They were prepared according to formerly reported protocols as referred. The initial activity of the precipitate was measured against the principal indicator E. avium EA5—our strain (Ent M, Ent 2019, Ent 9296—25,600 AU/mL, EntA/P, Ent 55—51,200 AU/mL, Ent 412—204,800 AU/mL, ED26E/7 Ent4231—6400 AU/mL, and EM41—800 AU/mL).
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Lauková, A.; Kandričáková, A.; Ščerbová, J.; Pogány Simonová, M.; Žitňan, R. Gram-Negative Microbiota Derived from Trout Fished in Slovakian Water Sources and Their Relationship to Postbiotics. Pathogens 2025, 14, 644. https://doi.org/10.3390/pathogens14070644

AMA Style

Lauková A, Kandričáková A, Ščerbová J, Pogány Simonová M, Žitňan R. Gram-Negative Microbiota Derived from Trout Fished in Slovakian Water Sources and Their Relationship to Postbiotics. Pathogens. 2025; 14(7):644. https://doi.org/10.3390/pathogens14070644

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Lauková, Andrea, Anna Kandričáková, Jana Ščerbová, Monika Pogány Simonová, and Rudolf Žitňan. 2025. "Gram-Negative Microbiota Derived from Trout Fished in Slovakian Water Sources and Their Relationship to Postbiotics" Pathogens 14, no. 7: 644. https://doi.org/10.3390/pathogens14070644

APA Style

Lauková, A., Kandričáková, A., Ščerbová, J., Pogány Simonová, M., & Žitňan, R. (2025). Gram-Negative Microbiota Derived from Trout Fished in Slovakian Water Sources and Their Relationship to Postbiotics. Pathogens, 14(7), 644. https://doi.org/10.3390/pathogens14070644

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