Microbiological identification of some culturable native strains associated with the digestive tract in Panaque cochliodon

Objective. To characterize the native strains associated with the intestinal tract of the species Panaque cochliodon by microbiology . Materials and methods. Three adult specimens were used. These were captured in the Magdalena River, transported, and sacrificed under animal welfare regulations. Dissection of the intestinal tract was performed, obtaining samples for microbiological isolation, using culture media, purifying the microorganisms, and performing metabolic biochemical tests and API 20E (Biomeriux) tests for their identification. Results. Information on the microbial population structure was obtained, reporting the phylum Proteobacteria with the species Pantoea sp ., Erwinia sp., Providencia stuarti, Providencia alcalifaciens, Serratia ficaria, Citrobacter koseri, and the phylum Firmicutes with the species Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus mycoides, Bacillus coagulans, and Bacillus circulans. Conclusions. The predominant culturable microorganisms in P. cohliodon belong to the phylum Proteobacteria and the phylum Firmicutes.


INTRODUCTION
Panaque cochliodon, the blue-eyed plec, is a fish endemic to the Cauca and Magdalena rivers reported as vulnerable (A2d) (1).It is in high demand in the ornamental fish market, and there is little knowledge of aspects related to its biology, intestinal microbiome, and digestive physiology.It has xylivorous habits (2), i.e., it consumes raw material of little nutritional value, such as lignin, but it is an important factor for this species to obtain energy and maintain its metabolic processes (3).Thus, microorganisms associated with the digestive system play a crucial role in digestion and metabolic modulation through the decomposition and assimilation of nutrients in the digestive system.These microorganisms also influence fish metabolism, participating in the synthesis of vitamins, the production of enzymes, and the regulation of key metabolic processes.Understanding how the microbiota interacts with the physiology and nutrition of fish is essential to optimize their diet (3)they present an intriguing system in which to examine the influence of host species identity on the vertebrate gut microbiome as well as to determine the potential role of gut bacteria in wood digestion.We characterized the gut microbiome of two co-occurring catfish genera and four species: Panaqolus albomaculatus, Panaqolus gnomus, Panaqolus nocturnus, and Panaque bathyphilus, as well as that of submerged wood on which they feed.The gut bacterial community did not significantly vary across three gut regions (proximal, mid, distal, defend themselves against pathogens (4), and stimulate the immune system of the host (4).Since their ontogeny, the microbiota has established interactions with fish; thus, the microorganisms colonize their body and establish symbiotic relationships that are key to their healthy development (5).Studies demonstrate a high diversity of microorganisms in freshwater fish, including some Gram-negative bacilli and some genera of the Enterobacteriaceae and Firmicutes families (6).
The microbiome of fish is gaining importance thanks to different studies that approach the structure of their communities; others relate diets with or without the inclusion of probiotics (7,8) or even organic acids, seeking to modulate pathogenic bacteria, preventing diseases and avoiding the use of antibiotics (8).Among the techniques for characterizing the microbiota, molecular ones (8) are found, in addition to biochemical tests, which allow identifying the genus and species of microorganisms (9).The biochemical characterization of microbial isolates allows determining their diversity and making inferences about their interactions (9,10).
Considering that Panaque cochliodon has no previous studies on intestinal microbiota, the aim of this study is to characterize the native strains associated with the intestinal tract of this species by microbiology.Dissection and isolation of the intestine.The euthanasia of the specimens was carried out with an overdose of Eugenol ® (200 mg/L) with subsequent spinal cord cutting to obtain the biological material (entrails).Then, it was cleaned with 70% v/v ethanol to reduce external contamination.Dissection was performed in the ventral area with a sterile scalpel, cutting from the anus to the mouth and extracting the entire intestine.

MATERIALS AND METHODS
Bacterial isolation.From the samples, 3 g of intestine per dissected fish were selected and incubated in BHI brain-heart broth for 2 h at 30°C under anaerobic conditions.After 2 h, the intestine was macerated (9,10), and successive dilutions were made until 10 -5 .Surface sowings of the 10 -3 , 10 -4 , and 10 -5 dilutions were performed in Agar BPLS (Bright green Phenol-Lactose-Sucrose red) (MERCK, 110747), Agar MacCONKEY (Merck, 100205), Agar (MAN, ROGOSA, and SHARPE) (Merck, 110660), nutrient Agar (Merck, 111471), and Agar M17 (Merck, 115108).The inoculated media were incubated at 30±2°C for 48 h.The 10 -2 dilution was heated at 80°C for 10 min and then cultured on nutrient agar to isolate Bacillus sp.After bacterial growth, the colony count was carried out (in CFU/g).The dilutions selected were those in which between 1 and 250 colonies were counted.The data obtained are expressed in Figure 1.Subsequently, the morphological identification of the colonies was carried out by Gram staining, and then the purification of each isolate that presented different particular traits was carried out.The isolates were coded and preserved at -80°C in Eppendorf tubes with nutrient broth plus glycerol until biochemical tests (9,10) were performed.
Gram-negative bacilli were identified using the API 20 E Biomeriux system (9,10).For this, the inoculum was standardized through the McFarland pattern (1.5x10 8 CFU/mL) and the protocols proposed by Puello-Caballero et al (9) and Gutiérrez-Ramírez et al (10).The culture media Agar BPLS (Bright green Phenol-Lactose-Sucrose red) (MERCK, 110747) and Agar MacConkey (Merck, 100205) were used for 24 h to obtain new and fresh cultures.Subsequently, each test was incubated with the inoculum according to the manufacturer's instructions.The reading was done to obtain the numerical profile that was assessed by the API WEB software (France, biomerieux.com),which showed an identification percentage of the isolates higher than 95% (9,10).All tests were carried out in the microbiology laboratory of Unilasallista.

RESULTS
Intestinal bacterial counts of P. cochliodon are shown in Figure 1.A higher number of Grampositive bacilli were recovered with a count of 9.9*10 5 CFU/g and Gram-positive cocci with 2.8*10 5 CFU/g.On the other hand, the count for lactic acid bacteria was lower, with a value of 2*10 4 CFU/g.These results follow the same trend in each dilution, registering a count of 1.5*10 6 CFU/g for Gram-negative bacilli and 9.9*10 5 CFU/g for Gram-positives.Observing the colonies allowed the determination of the typical and relevant morphological characteristics of the isolate, associated with the shape, texture, elevation, and color, among others, which, together with its response to the Gram stain, allowed an approach to its classification.In this sense, irregular shapes, wavy and lobed edges, and various types of elevations are shown in Figures 2, 3, and 4.

4a 4b 2a 2b
For Gram-negative bacilli, the API 20E system indicated the predominance of the order Enterobacterales, with four different families: Enterobacteriaceae, Erwinaceae, Morganellaceae, and Yersinaceae.The genera and species are indicated in Table 1, with their respective coding based on the isolation records.
The enzymatic activity for the six intestinal isolates of P. cochliodon that gave Grampositive staining was evaluated in selective media (Table 2).The activity of the enzymes catalase, caseinase, amylase, and lecithinase was determined, finding that all the isolates have the enzyme catalase and caseinase; three of them have the enzyme lecithinase and four have the enzyme amylase.This could be observed in the specific culture media and with the reagent hydrogen peroxide (H 2 O 2 ) for catalase determination.

DISCUSSION
Considering that fish are the group with the greatest taxonomic and ecological diversity of vertebrates, knowledge of their microbiome is still to be resolved (11).This is probably due to its high complexity, which implies greater challenges in understanding its structure and function (12).Currently, the development of molecular techniques is frequently used in studies based on culture media; these techniques have allowed inferences about the function of fish microbiota (12,13).However, the sensitivity of the microorganism and the tests must be considered when describing and classifying their microbiome (13,14).For P. cochliodon, this is the first report of cultured native strains associated with their gastrointestinal tract, understanding that it has been reported that the capture and confinement of wild fish can influence the structure of the microbiota ( 14), in addition to other aspects such as the environment, phylogeny, host genetics and diets (14,15).It has generally been permitted that the quantity of bacterial microorganisms in the digestive system of fish can be higher than 10 8 heterotrophs/g and 10 5 anaerobes/g (15,16).In the current study, the values found were higher than 10 5 , indicating a large number of microorganisms recovered.
The sporulated bacilli are likely to be present in higher quantities due to their metabolic dynamics, generating important compound hydrolysis functions (16).
It is known that the main groups of bacteria that colonize the gastrointestinal tract in fish are aerobes, facultative anaerobes, and obligate anaerobes (17,18) and, specifically, in freshwater fish, Aeromonas, Pseudomonas, and Bacteroides type A predominate together with Plesiomonas; the least abundant groups are Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, Bacteroides type B, and Fusarium (18,19).In the current study, populations of the order Enterobacterales were found, whose characteristics are associated with fermentation processes of glucose and other carbohydrates (20), although they are also associated with diseases due to their opportunistic nature (20,21).Similar findings have been reported for the species Piaractus brachypomus (21) and other freshwater species but with molecular methods for this same phylum, which include some genera reported in the current study: Oncorhynchus mykiss (22), Megalograma amblycephala, Ctenopharyngdon idellus, Carassius auratus, Ciprinus carpio, Hypophthalmichthys nobilis and H. molitrix (22), Oreochromis niloticus (22), Ictalurus punctatus (22), and Danio rerio (23).
By understanding the origin of the captures, it is expected that influences of anthropic origin and the environment can determine the presence of these groups in P. cochliodon, as has been shown in other studies (23).On the other hand, the particular eating habit of P. cochliodon could provide information about the presence of these types of microorganisms and their importance in plant matter transformation to obtain energy.In this regard, mechanisms have been elucidated by which many wood eaters such as Panaque nigrolineatus and Hypostomus pyrineusi use the biofilm that is associated with wood as a source of microorganisms with cellulolytic activity, being part of the natural detritivore or xylivorous diet (24).Conversely, work with isotopes has shown higher biotransforming activity of the organisms associated with the biofilm compared to that of endosymbiotic microorganisms (24), which has been corroborated for other wood eaters of the genera Panaqolus and Panaque, suggesting a greater role for the environmental microbiome (wood and biofilm) than for the intestinal microbiome (24,25), including other fish with different eating habits (25).
Another phylum of importance in fish is Firmicutes, which, together with Proteobacteria and Bacteroidetes, represent more than 90% of the intestinal microbiota (25).In P. cochliodon, Firmicutes was also found.Similar findings were reported for other fish in the same environment but with different feeding habits (25,26).Within Firmicutes, the genus Bacillus is found, which is Gram-positive, catalase-positive, forms endospores, is aerobic or facultative anaerobic, and is highly ubiquitous in nature (26), including aquatic environments (26)  Ictalurus punctatus (29).This species has also been reported for Salmo salar (30) and some representatives of the families Cyprinidae and Cichlidae (31).In addition, cellulolytic activity has been demonstrated in fish intestine isolates from the Amazon River (Piaractus brachypomus and Leporinus friderici) (32).Bacillus coagulans has been reported for Catla catla, Labeo rohita, and Cirrinhus mrigala, indicating the activity of the enzymes amylase, protease, and cellulase (32).In turn, B. circulans has been discovered in the intestine of Cyprinus carpio and Oreochromis mossambicus with high cellulolytic activity (33); furthermore, also for Ctenopharyngodon Idella, with the same enzymatic activity (33).In the species Salmo salar, B. thuringiensis is reported, indicating a broad spectrum of enzymatic activity (34).
On the other hand, the ability to produce spores means that Bacillus species can occupy various habitats, offering important characteristics in the bio-remediation of industrial wastewater (35,36), in the care for water quality in aquaculture (37), in sustainable aquaculture (37) and has been widely used as probiotics in different animal models (37,38).In fish, its application has direct implications for improving growth and feed conversion (38), stress resistance (38), immunostimulation, disease resistance (39), and, overall, their well-being (39). In

Figure 1 .
Figure 1.Count in logarithm of colony forming units (CFU/g) of the intestinal microbiome of Panaque cochliodon.

Figure 3 .
Figure 3. Morphological identification of the colony and bacterial cell staining.BPLS agar culture medium;observation of a colony with an irregular shape, wavy edge, and soft consistency (3a).Gram-negative bacilli (3b), 100x magnification.

Table 1 .
Bacterial cultures selected and identified with the API 20E system.

Table 2 .
Biochemical characteristics of some bacteria of the genus Bacillus.