MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY BASED IDENTIFICATION OF THE FISH GUT MICROBIOTA

Back to full issue:
August – September 2019, vol. 9, no. 1
pages: 151-155
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2019.9.1.151-155
Abstract: The aim of the study was to investigate the composition of intestinal microflora of freshwater fish in Latvia. A total amount of 28 fish were collected from fishermen (n=20) and retail outlets (n=8), including roach Rutilus rutilus, n=15, crucian carp Carassius carassius, n=5, perch Perca fluvialitis, n=5, bream Blicca bjoerkna, n=3. Microbiological testing consisted of the detection of total viable count (TPC), Enterobacteriaceae and coliforms with subsequent identification with MALDI-TOF Biotyper. TPC, coliforms and Enterobacteriaceae counts ranged from 2.7±0.4 to 5.4±0.3, 2.4±1.5 to 3.7±0.7 and 2.00±1.2 to 3.7±2.5 log cfu/g in gut of wild crucian carp and retailed roach, wild perch and retailed roach, and crucian carp and bream, accordingly. The TPC, coliforms and Enterobacteriaceae counts were significantly higher in retailed fish than in wild fish gut samples (P≤0.05). Gut microbiota were represented by Proteobacteria (93.0%), Firmicutes (3.9%) and Ascomycota (3.1%). The most abundant families were Enterobacteriacea (50.8%) and Pseudomonadaceae (36.7%). Rahnella aquatilis, Serratia fonticola and Pantoe aagglomerans were the most abundant among Enterobacteriaceae while Pseudomonas extremorientalis and P. fragi among the Pseudomonadaceae. Results of the present study show that the gut of freshwater fish were mostly represented by Enterobacteriaceae and Pseudomonadaceae and the presence of fish bacterial pathogens must be considered.
XMLs: | NLM DTD xml | Copernicus xml |
Full text pdf download link: Issue navigation: August – September 2019, vol. 9, no. 1:
prev. article |p. 144-150| next article |p. 156-159|
Fulltext:

MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY BASED IDENTIFICATION OF THE FISH GUT MICROBIOTA


AUTHORS

Miroslava Kačániová, Margarita Terentjeva, Alīna Klūga, Monika Březinová, Miriam Kádasi Horáková

ABSTRACT

The aim of the study was to investigate the composition of intestinal microflora of freshwater fish in Latvia. A total amount of 28 fish were collected from fishermen (n=20) and retail outlets (n=8), including roach Rutilus rutilus, n=15, crucian carp Carassius carassius, n=5, perch Perca fluvialitis, n=5, bream Blicca bjoerkna, n=3. Microbiological testing consisted of the detection of total viable count (TPC), Enterobacteriaceae and coliforms with subsequent identification with MALDI-TOF Biotyper. TPC, coliforms and Enterobacteriaceae counts ranged from 2.7±0.4 to 5.4±0.3, 2.4±1.5 to 3.7±0.7 and 2.00±1.2 to 3.7±2.5 log cfu/g in gut of wild crucian carp and retailed roach, wild perch and retailed roach, and crucian carp and bream, accordingly. The TPC, coliforms and Enterobacteriaceae counts were significantly higher in retailed fish than in wild fish gut samples (P≤0.05). Gut microbiota were represented by Proteobacteria (93.0%), Firmicutes (3.9%) and Ascomycota (3.1%). The most abundant families were Enterobacteriacea (50.8%) and Pseudomonadaceae (36.7%). Rahnella aquatilis, Serratia fonticola and Pantoe aagglomerans were the most abundant among Enterobacteriaceae while Pseudomonas extremorientalis and P. fragi among the Pseudomonadaceae. Results of the present study show that the gut of freshwater fish were mostly represented by Enterobacteriaceae and Pseudomonadaceae and the presence of fish bacterial pathogens must be considered.


KEYWORDS

bream, crucian carp, MALDI-TOF MS Biotyper, Enterobacteriaceae, Pseudomonadaceae, roach

INTRODUCTION

The digestive tract of fish is a habitat of heterogeneous microflora and is colonized by a high variety and number of microorganisms (Burr et al., 2005). The existing symbiosis between host and the intestinal microflora is of great significance for all live organisms, including fish (Sugita et al., 1997). In fish, the gut microbiota is important for digestion of food, protection of fish against the bacterial pathogens and development of immunological response. Impairment in balance of gut microbiota affects the fish health, consequently, the stability and composition of gut microbiota are important (Gómez and Balcázar, 2008).

Composition of fish gut microbiota depends on various influencing factors, including fish species, age, nutritional, genetic factors and environmental conditions of habitat (Gómez and Balcázar, 2008; Floris et al., 2013). Differences between the composition of intestinal microbiota of marine and freshwater fish were identified and the freshwater fish gut microorganisms were more diverse with the genera Aeromonas, Flavobacterium, Pseudomonas and the family Enterobacteriaceae were found to be dominant (Skrodenyté-Arbačiauskiené, 2008). Composition of gut microbiota may differ between the individual fish and fish species (González et al., 1999).

Since the gut microbiota consist of various groups of microorganisms of different functional significance, an establishment of balanced gut microflora is essential. The normal indigenous microbiota act competitively and prevent the colonization of gut by pathogens, however, the different other groups of pathogenic microorganisms of fish, animal and human health significance can be found in gut (Austin, 2006). Thus, the identification and analysis of fish gut microbiota helps not only recognize the composition of fish microflora in different environments, but also to tackle potentially pathogenic microorganisms affecting the fish health. Fish is an important food source and the studies on fish gut microbiota are important for an assessment of fish health and safety of fish for consumption as well (Holben et al., 2002).

Freshwater fish is a significant part of fish fauna in Latvia and usually the most accessible for fishermen. Of total more than 40 freshwater species represented, roach Rutilus rutilus, perch Perca fluviatilis and bream Blicca bjoerkna were found to be the main species caught by fishermen in inland waters (Birzaks, 2008).

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged in the recent years as a reliable and rapid method for identification and characterization of microorganisms. The MALDI-TOF MS has several advantages in comparison with microbiological method in terms of sensitivity and economical aspects, including processing, application and staff costs. The applications of the MALDI-TOF for identification of microorganisms, detection of food-borne pathogens, strain typing, characterization of antibiotic resistance, epidemiological studies etc. were described (Singhal et al., 2015). The studies on the characterization of freshwater fish microbiota with MALFI-TOF MS are very limited.

The aim of the present study was to identify and analyse the freshwater fish gut microbiota with MALDI-TOF MS Biotyper.

MATERIAL AND METHODS

Sampling

Altogether, an amount of 28 freshwater fish were collected in Latvia. Samples included roach (Rutilus rutilus n=15), crucian carp (Carassius carassius, n=5), perch (Perca fluviatilis, n=5) and bream (Blicca bjoerkna, n=3). Samples were obtained from fishermen immediately after angling (n=20) and purchased at retail market (n=8). Fish from retail market were purchased as a whole, ungutted fish. Sampled fish were placed on ice and transported to the laboratory for microbiological testing. Examination of fish was initiated within 2 h after sampling and the gut was aseptically removed from surrounding tissues and used for further investigations.

Microbiological testing of gut content

For microbiological testing, the total plate count (TPC), Enterobacteriaceae and total coliforms were detected. An amount of 1 g of the gut content was transferred to 9 mL of peptone buffered water (Oxoid, Basingstoke, United Kingdom) and mixed to obtain the initial dilution. The initial dilution was used for the preparation of further serial dilutions. Then, a quantity of 1 mL of the initial and serial dilutions was transferred in sterile Petri dishes and covered with 15 mL of molten Plate Count Agar (PCA) for TPC, Violet Red Bile Glucose Agar (VRBGA) for Enterobacteriaceae and Violet Red Bile Lactose Agar (VRBLA) for total coliforms (Oxoid). After inoculation, the PCA plates were incubated at 30°C for 72h, but VRBGA and VRBLA at 37°C for 24h with evaluation of bacterial growth after incubation. All colonies were counted on PCA, while the typical Enterobacteriaceae and coliform colonies were enumerated on VRBGA and VRBLA. Altogether, one to twenty five colonies were selected from the each plate for further confirmation with MALDI-TOF.

Identification of bacteria with MALDI-TOF MS Biotyper

Altogether, 128 microbial colonies were used for confirmation and the colonies were picked up from agar, suspended in 300 μl of sterile distilled water and mixed thoroughly. Then, an amount of 900 μL of absolute ethanol (99%, Sigma-Aldrich, USA) was added. The mixture was centrifuged at 13 000 x g for 2 min. Later, the supernatant was removed and the pellet was centrifuged. Residual ethanol was completely removed by pipetting and the pellet was allowed to dry at a room temperature. Subsequently, an amount of 10 μL of formic acid (70%, Sigma-Aldrich, USA) was added to the pellet and mixed with a sterile toothpick. Next, a 10 μL of acetonitrile (100%, Sigma-Aldrich, USA) was added and mixed thoroughly. The solution was centrifuged at maximum speed for 2 min and 1 μL of the supernatant was spotted on a polished MALDI target plate (Bruker Daltonics, Germany). Immediately after drying a 1 μL of the matrix solution was added to each spot and allowed to air dry. The matrix used was a saturated solution of HCCA: α-cyano-4-hydroxycinnamic acid (Bruker Daltonics, Germany) dissolved in 50 % acetonitrile with 0.025 % trifluoroacetic acid (TFA) (100%, Sigma-Aldrich, USA). The matrix solution preparation (2.5 mg of HCCA) contains 500 µL of acetonitrile, 475 µL of ultrapure water and 25 µL of trifluoroacetic acid. An amount of 250 µL of the solution was added to the 2.5 mg of HCCA. Samples were processed in the MALDI-TOF MS (Microflex LT/SH, Bruker Daltonics, Germany) with flex Control software and results were obtained with Realtime Classification software (RTC) (Bruker Daltonics, Germany) (Kačániová et al., 2019).

Statistical analyses

All bacterial counts were transferred into a decimal log. The One-Way ANOVA test was used for calculation of significance of differences between bacterial counts in different fish species.

RESULTS

Total plate count (TPC) ranged from 2.7±0.5 to 5.4±0.3 log cfu/g in the gut of wild crucian carp and retailed roach, respectively. Coliform counts were from 2.4±1.5 to 3.7±0.7 log cfu.g-1 in wild perch and retailed roach, but Enterobacteriaceae from 2.00±1.2 to 3.7±2.5 log cfu.g-1 in the gut of crucian carp and bream, accordingly (Table 1). The TPC, coliforms and Enterobacteriaceae counts were significantly higher in retailed fish gut than in wild fish gut samples (P≤0.05), while the significant differences between the bacterial counts of retailed fish were not identified (P≥0.05).

Table 1 Total bacterial count, Enterobacteriaceae and coliform counts in gut freshwater fish (in log cfu/g)

Species Origin No. of samples TPC Coliforms Enterobacteriaceae
Roach Wild 10 4.39±0.57a 2.96±0.46b 1.97±1.41
Retailed 5 5.39±0.29a 3.69±0.69b 3.7±0.65
Crucian carp Wild 5 2.72±0.46a 2.55±0.36b 1.95±1.15
Perch Wild 5 4.15±0.53a 2.39±1.48b 2.74±0.61
Bream Retailed 3 5.3±0.29a 3.62±2.35b 3.72±2.49

a differences between TPC in wild roach and crucian carp were significant (P<0.05), while there were no significant differences between TPC in retailed roach and bream gut (P>0.05)

b differences in coliform counts were not significant between wild (roach, crucian carp, perch) and retailed fish (roach, bream) (P>0.05)

c differences in Enterobacteriaceae counts between wild (roach, crucian carp, perch) and retailed fish (roach, bream) were significant (P<0.05)

The most abundant microbial phylum of fish gut was Proteobacteria (93.0%) followed by Firmicutes (3.9%) and Ascomycota (3.1%). The most abundant microbial families were Enterobacteriaceae (50.8%) and Pseudomonadaceae (36.7%) while the less abundant were Bacillaceae, Lactobacillaceae, Peptostreptococcaceae, Sphingomonadaceae and Xanthobactereaceae (0.8% each). Enterobacteriaceae was the predominant in gut of the wild roach (57.4%), crucian carp (50%), but there were no differences between the abundance of Enterobacteriaceae and Pseudomonadaceae in retailed roach and perch intestinal samples (P>0.05). The families Aeromonadaceae and Clostridiaceae were the most abundant in bream gut (33.3% each) (Table 2). The most diverse microbiota were recovered from the wild roach gut, but the less diverse from retailed roach gut with six and two phyla were identified, respectively.

Table 2 Abundance of microorganisms in freshwater fish gut

Family Wild roach Rutilus rutilus Roach retailed Rutilus rutilus Crucuan carp Carassius carassius Perch

Perca fluvialitis

Bream

Blicca bjoerkna

                                      No. of isolates (%)
Aeromonadaceae 0 (0) 0 (0) 2 (5.9) 0 (0) 2 (33.3)
Bacillaceae 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Clostridiaceae 0 (0) 0 (0) 0 (0) 0 (0) 2 (33.3)
Enterobacteriaceae 35 (57.4) 7 (50.0) 17 (50.0) 5 (38.6) 1 (16.7)
Lactobacillaceae 0 (0) 0 (0) 0 (0) 0 (0) 1 (16.7)
Peptostreptococcaceae 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Pseudomonadaceae 22 (36.2) 7 (50.0) 13 (38.2) 5 (38.6) 0 (0)
Sphingomonadaceae 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Streptococcaceae 0 (0) 0 (0) 0 (0) 1 (7.6) 0 (0)
Xanthobactereaceae 0 (0) 0 (0) 0 (0) 1 (7.6) 0 (0)
Saccharomycetaceae 1.6 (0) 0 (0) 2 (5.9) 1 (7.6) 0 (0)
Total 61 (100) 14 (100) 34 (100) 13 (100) 6 (100)

Among the Enterobacteriaceae, the most abundant bacterial species were Rahnella aquatilis (17 isolates), Serratia fonticola (9 isolates) and Pantoea aagglomerans (8 isolates). Beside the Pseudomonadaceae, Pseudomonas extremorientalis and P. fragi were the most abundant (6 isolates).

Rahnella aquatilis and Providencia heimbachae were the most abundant Enterobacteriaceae in wild (19.67%) and retail roach gut (21.43%), respectively. Pantoea agglomerans and Rahnella aquatilis were the most abundant in crucian carp gut (29.4%) but Enterobacter cloacae and Enterobacter amnigenus in perch and bream gut (40%) and one isolate (100%), respectively (Table 3).

Table 3 Microflora of gut of wild and retailed freshwater fish

Roach Crucian carp Perch Bream
Family Identified species Wild Retailed Wild Wild Retailed
N=10 N=5 (n=5) (n=5) (n=3)
No. of isolates identified (%)
Aeromonadaceae A. bestiarum 0 (0) 0 (0) 1 (2.9) 0 (0) 0 (0)
  Aeromonas eucrenophila 0 (0) 0 (0) 0 (0) 0 (0) 1 (12.5)
  Aeromonas hydrophila 0 (0) 0 (0) 0 (0) 0 (0) 1 (50)
  Aeromonas veronii 0 (0) 0 (0) 1 (2.9) 0 (0) 0 (0)
Bacillaceae Bacillus megaterium 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Clostridiaceae Clostridium septicum 0 (0) 0 (0) 0 (0) 0 (0) 1 (12.5)
  Clostridium chauvoei 0 (0) 0 (0) 0 (0) 0 (0) 1 (12.5)
Enterobacteriaceae Buttiauxella ferragutiae 0 (0) 0(0) 3 (8.7) 0 (0) 0 (0)
  Citrobacter gillenii 2 (3.3) 0 (0) 0 (0) 0 (0) 0 (0)
  Enterobacter amnigenus 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Enterobacter cloacae 2 (3.3) 0 (0) 0 (0) 2 (15.3) 0 (0)
  Ewingella americana 3 (4.9) 0 (0) 0 (0) 0 (0) 0 (0)
  Hafnia alvei 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Klebsiella oxytoca 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
  Enterobacter amnigenus 1 (1.6) 0 (0) 0 (0) 0 (0) 1 (12.5)
  Moellerella wisconsensis 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pantoea agglomerans 2 (3.3) 0 (0) 5 (14.8) 1 (7.7) 0 (0)
  Pluralibacter pyrinus 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Providencia heimbachae 0 (0) 3 (21.6) 0 (0) 0 (0) 0 (0)
  Rahnella aquatilis 12 (19.6) 2 (14.2) 5 (14.8) 0 (0) 1 (12.5)
  Raoultella ornithinolytica 0 (0) 0 (0) 0 (0) 1 (20) 0 (0)
  Serratia entomophila 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Serratia fonticola 4 (7.3) 1 (7.1) 4 (11.6) 0 (0) 0 (0)
  Serratia liquefaciens 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Serratia plymuthica 2 (3.3) 0 (0) 0 (0) 0 (0) 0 (0)
  Yersinia intermedia 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Yersinia ruckeri 0 (0) 1 (7.1) 0 (0) 0 (0) 0 (0)
Lactobacillaceae Lactobacillus mucosae 0 (0) 0 (0) 0 (0) 0 (0) 1 (12.5)
Peptostreptococcaceae Filifactor villosus 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Pseudomonadaceae Pseudomonas antarctica 1 (1.6) 0 (0) 0 (0) 1 (20) 0 (0)
  Pseudomonas brenneri 0 (0) 0 (0) 1 (2.9) 0 (0) 0 (0)
  Pseudomonas extremorientalis 2 (3.3) 0 (0) 4 (11.6) 0 (0) 0 (0)
  Pseudomonas fluorescens 2 (3.3) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas frederiksbergensis 2 (3.3) 0 (0) 2 (6) 0 (0) 0 (0)
  Pseudomonas fragi 3(4.9) 3 (21.6) 0 (0) 0 (0) 1 (12.5)
  Pseudomonas fulva 0 (0) 0 (0) 1 (2.9) 0 (0) 0 (0)
  Pseudomonas gessardii 1 (1.6) 0 (0) 1 (2.9) 0 (0) 0 (0)
  Pseudomonas grimontii 1 (1.6) 0 (0) 0 (0) 1 (20.0) 0 (0)
  Pseudomonas koreensis 0 (0) 2 (14.2) 2 (6) 0 (0) 0 (0)
  Pseudomonas libanensis 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas lundensis 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas marginalis 1 (1.6) 0 (0) 2 (6) 1 (20.0) 0 (0)
  Pseudomonas orientalis 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas proteolytica 2 (3.3) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas putida 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas rhodesiae 0 (0) 1 (7.1) 0 (0) 0 (0) 0 (0)
  Pseudomonas synxantha 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
  Pseudomonas thivervalensis 0 (0) 1 (7.1) 0 (0) 0 (0) 0 (0)
  Pseudomonas tolaasii 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
  Presudomonas trivialis 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
  Pseudomonas veronii 2 (3.3) 0 (0) 0 (0) 0 (0) 0 (0)
Sphingomonadaceae Sphingopyxis terrae 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0)
Streptococcaceae Streptococcus salivarius 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
Saccharomycetaceae Candida pelliculosa 1 (1.6) 0 (0) 2 (6) 0 (0) 0 (0)
  Pichia fermentans 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
Xanthobacteraceae Starkeya novella 0 (0) 0 (0) 0 (0) 1 (7.7) 0 (0)
Total   61 (100) 14 (100) 34 (100) 13 (100) 6 (100)

DISCUSSION

The TPC, Enterobacteriaceae and coliform counts identified in the present study were in agreement with the previously reported (Navarrete et al., 2010; Wu et al., 2010; Floris, 2013; Kluga et al., 2017). The bacterial counts in gut depend on environmental factors, water and feed quality and diet. Bacterial counts can vary between the individual fish but, in general, the gut is inhabited by a large number of microorganisms (Spanggaard et al., 2000). Bacterial counts in retailed fish were higher than in freshly caught fish that could be attributable to storage on ice before the sale, as it is known the bacteria may proliferate during the storage and impair quality of the fish product (Al Bulushi et al., 2008). Our results on the bacterial counts of freshly caught freshwater fish correspond to findings in fish gut originated from cold and relatively unpolluted waters (González et al., 1999).

A phylum Proteobacteria was found to be predominating in gut of all studies freshwater fish species. Bacteria representing the Proteobacteria and the Firmicutes phylum were frequently reported to be present in fish gut. Proteobacteria was the predominated phylum in the gut of yellow catfish (Pelteobagrus fulvidraco), gilthead sea bream (Sparus aurata) and crucian carp Carassius gibelio (Wu et al., 2010; Floris, 2013; Kashinskaya et al., 2015). The results of our study support the observation that Proteobacteria were prevalent in the gut of freshwater fish (Kashinskaya et al., 2015).

Enterobacteriaceae family was predominated in the gut of wild roach and crucian carp, while there were no differences between the abundance of Enterobacteriaceae and Pseudomonas in the gut of retailed roach, bream and perch. In general, our findings are in line with the previously reported and Enterobacteriaceae was found to be predominated in freshwater salmon (Salmo salar) and sea trout (Salmo trutta trutta) (Skrodenyté-Arbačiauskiené et al., 2008). Enterobacteriaceae and Pseudomonas were found to be the most abundant in the gut of roach (Rutilus rutilus) (Skrodenyté-Arbačiauskiené, 2007). In contrast, Floris et al. (2013) reported that Pseudomonas spp. were predominant in gilthead sea bream (Sparus aurata) in two coastal lagoons of Sardinia. Pseudomonas are metabolically versatile microorganisms ubiquitous in the environment and frequently associated with fish and water as a habitat of fish (Vaz-Moreira et al., 2012). Results of our study show that Pseudomonas spp. alongside with Enterobacteriaceae are the important representatives of the intestinal microbiota of wild freshwater fish. Rahnella aquatilis, Serratia fonticola and Pantoea agglomerans of the Enterobacteriaceae family were found be the most abundant that is in agreement with the previous studies on broad distribution of the bacteria in the environment, including the fish. The bacteria were identified in water, soil, plants, snails, slug, molluscs and the intestinal tract of fish (Derlet and Carlson, 2004; Piotrowska-Seget et al., 2005; Skrodenyté-Arbačiauskiené, 2007).

Other microorganisms as Bacillus, Buttiauxella, Ewingella, Serratia, Providencia, Raoultella, Sphingomonas, Candida, Pichia and Starkeya were associated with water, soil, vegetables, foods, insects, plants and trees (White et al., 1996; Kelly et al., 2000; Hurst and Jackson, 2002; Barchiesi et al., 2005; Aravind et al., 2009; Vadkertiová et al., 2012). Additionally, C. gillenii and M. wisconsensis were recognized as the members of fish microflora in previous studies (Skrodenyté-Arbačiauskiené et al., 2008; Lü et al., 2011). Due to wide distribution of the microorganisms in the environment, they could enter the intestinal tract of fish. Our findings revealed that the gut of freshwater fish may be a habitat of those microorganisms.

Pseudomonas spp. were frequently isolated from fish and are recognized to be the specific spoilage microorganism involved in the deterioration of the quality of freshly chilled fish (Gram and Dalgaard, 2002). Since the Pseudomonas spp. may develop the rapid growth in favourable condition, the predominance of Pseudomonas spp. in fish is undesirable and lead to the fish spoilage. P. fragi, P. lundensis and P. fluorescens were found to be the predominated in the fish at the end of shelf-life and contributed the spoilage (Tryfinopoulou et al., 2002). Thus, the abundance of Pseudomonas in the gut may results in additional contamination of fish fillet during the gutting and predisposes the bacterial spoilage processes (MacMillan and Santucci, 1990).

Pseudomonas were associated with fish diseases and fish pathogenic Pseudomonas spp. identified in the present study. P. fluorescens was the causative agent of bacterial haemorrhagic septicaemia in rainbow trout, carp and chronic disease in catfish (Shahi and Mallik, 2014). P. koreensis caused the eye infection in golden mahseer in India (Shahi and Mallik, 2014), but P. putida ulcers in rainbow trout (Altinok et al., 2006). Alongside with the Pseudomonas spp., Hafnia alvei, Enterobacter cloaceae, Yersinia intermedia and Y. ruckeri were reported to be the fish pathogenic (Acosta et al., 2002; Toback et al., 2007; Sekar et al., 2008). Aeromonas are present primary in aquatic environments and A. hydrophila was found to inhabit normally the intestinal tract of fish (Carvalho et al., 2012). However, the bacteria may become the opportunistic fish pathogen in a variety of farmed fish during stressful growth conditions (Li et al., 2013). A. bestiarum and A. veronii are expected to be pathogenic for fish and were isolated from common carp and trout (Kozińska, 2007). Abovementioned bacteria and particularly Y. ruckeri may result in fish diseases with high mortality and attributed significant economic losses, therefore, the presence of those pathogens must be taken into consideration. The pathogenic microorganisms were isolated both from retail and wild fish, indicating the circulation of the pathogenic microorganisms in the environment and aquaculture.

Fish may carry the microorganisms which are opportunistic pathogens or pathogenic to consumers. Consumption of fish contaminated with A. hydrophila, A. caviae and A. veronii bv. sobria may cause the foodborne gastroenteritis. Aeromonas were linked to wound and respiratory infections, septicaemia, liver abscesses, urinary tract and eye infections. K. oxytoca, E. cloaceae, P. agglomerans, R. aquatilis and R. ornitholytica are nosocomial pathogens responsible for different clinical manifestations, including the urinary tract, respiratory tract, wound, skin and soft tissues infections and bacteremia. Clostridium spp. may result in gas gangrene with human and the animal patient became affected (Tash, 2005; Cruz et al., 2007; Gorkiewicz, 2009). Our findings indicate that fish may serve as a source of the microorganisms of fish and public health significance. The present results reveal the potential risks of bacterial contamination of fishes from Latvia. Periodic monitoring of microorganisms pathogenic for fish and consumers, is important to identify any potential treat (Alikunhi et al., 2016).

CONCLUSION

In conclusion, the present study confirms the predominance of Enterobacteriaceae and Pseudomonadaceae in of the gut freshwater fish. The composition of microbiota may alter the fish health alongside with the quality and safety of fish meat and fish products. The fish intestinal tract may serve as a habitat for microorganisms with fish and public health significance, therefore the results of present study indicate that fish may be an important vector for transmission of potentially pathogenic microorganisms for fish and fish consumers.

Acknowledgement: This work was supported by grant European Community project no. 26220220180: Building the Research Centre „AgroBioTech“ and by the grants APVV-15-0544.

REFERENCES

ACOSTA, F., REAL, F., CABALLERO, M. J., SIEIRO, C., FERNÁNDEZ, A., RODRÍGUEZ, L. A. 2002. Evaluation of immunohistochemical and microbiological methods for the diagnosis of brown trout infected with Hafnia alvei. Journal of Aquatic Animal Health, 14(1), 77-83. https://doi.org/10.1577/1548-8667(2002)014<0077:eoiamm>2.0.co;2

AL BULUSHI, I. M., POOLE, S., DEETH, H. C., DYKES, G. A. 2008. Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical fish. World Journal of Microbiology and Biotechnology, 24(9), 1867-1875. https://doi.org/10.1007/s11274-008-9687-5

ALIKUNHI, N. M., BATANG, Z. B., ALJAHDALI, H. A., AZIZ, M., & AL-SUWAILEM, A. M. 2016. Culture-dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing. Saudi Journal of Biological Sciences, 24(6), 1105–1116. https://doi.org/10.1016/j.sjbs.2016.05.017

ALTINOK, I., KAYIS, S., & CAPLIN, E. 2006. Ulcers caused by Pseudomonas putida infection in rainbow trout. Aquaculture, 261, 850-855. https://doi.org/10.1016/j.aquaculture.2006.09.009 

ARAVIND, R., KUMAR, A., EAPEN, S. J., RAMANA, K. V. 2009. Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification, and evaluation against Phytophthora. Letters in Applied Microbiology, 48(1), 58-64. https://doi.org/10.1111/j.1472-765x.2008.02486.x 

AUSTIN, B. 2006. The bacterial microflora of fish, revised. The Scientific World Journal, 6, 931-945. https://doi.org/10.1100/tsw.2006.181 

BARCHIESI, F., TORTORANO, A. M., FALCONI DI FRANCESCO, L., RIGONI, A., GIACOMETTI A., SPREGHINI, E., SCALISE, G., VIVIANI, M. A. 2005. Genotypic variation and antifungal susceptibilities of Candida pelliculosa clinical isolates. Journal of Medical Microbiology, 54(3), 279-285. https://doi.org/10.1099/jmm.0.45850-0 

BIRZAKS, J. 2008. Fish resources of inland waters of Latvia and their usage. In: E. Riekštiņš (Eds.), Latvian Fisheries:  Fisheries Collaboration Network, Rīga, pp. 66-82.

BURR, G., GATLIN, D., RICKE, S. 2005. Microbial ecology of the gastrointestinal tract of fish and the potential application of prebiotics and probiotics in finfish aquaculture. Journal of the World Aquaculture Society, 36(4), 425-435. https://doi.org/10.1111/j.1749-7345.2005.tb00390.x 

CARVALHO, M. J., MARTÍNEZ-MURCIA, A., ESTEVES, A. C., CORREIA, A., SAAVEDRA, M. J. 2012. Phylogenetic diversity, antibiotic resistance and virulence traits of Aeromonas spp. from untreated waters for human consumption. International Journal of Food Microbiology, 159(3), 230-239. https://doi.org/10.1016/j.ijfoodmicro.2012.09.008 

CRUZ, A. T., CAZACU, A. C., ALLEN, C. H. 2007. Pantoea agglomerans, a plant pathogen causing human disease. Journal of Clinical Microbiology, 45(6), 1989-1992. https://doi.org/10.1128/jcm.00632-07 

DERLET, R. W., CARLSON, J. R. 2004. An analysis of wilderness water in Kings Canyon, Sequoia, and Yosemite National Parks for coliforms and pathologic bacteria. Wilderness & Environmental Medicine, 15(4), 238-244. https://doi.org/10.1580/1080-6032(2004)015[0238:aaowwi]2.0.co;2 

FLORIS, R., MANCA, S., FOIS, N. 2013. Microbial ecology of intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758) from two coastal lagoons of Sardinia (Italy). Transitional Waters Bulletin, 7, 4-12.

GÓMEZ, G. D., BALCÁZAR, J. L. 2008. A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunology and Medical Microbiology, 52(2), 145-154. https://doi.org/10.1111/j.1574-695x.2007.00343.x 

GONZÁLEZ, C. J., LÓPEZ-DÍAZ, T. M., GARCÍA-LÓPEZ, M. L., PRIETO, M., OTERO, A. 1999. Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). Journal of Food Protection, 62(11), 1270-1277. https://doi.org/10.4315/0362-028x-62.11.1270 

GORKIEWICZ, G. 2009. Nosocomial and antibiotic-associated diarrhoea caused by organisms other than Clostridium difficile. International Journal of Antimicrobial Agents, 33, 37-41. https://doi.org/10.1016/s0924-8579(09)70015-9 

GRAM, L., DALGAARD, P. 2002. Fish spoilage bacteria-problems and solutions. Current Opinion in Biotechnology, 13(3), 62-266. https://doi.org/10.1016/s0958-1669(02)00309-9 

HOLBEN, W. E., WILLIAMS, P., SAARINEN, M., SÄRKILAHTI, L. K., APAJALAHTI, J. H. A. 2002. Phylogenetic analysis of intestinal microflora indicates a novel mycoplasma phylotype in farmed and wild salmon. Microbial Ecology, 44(2), 175-185. https://doi.org/10.1007/s00248-002-1011-6 

HURST, M. R. H., JACKSON, T. A. 2002. Use of the green fluorescent protein to monitor the fate of Serratia entomophila causing amber disease in the New Zealand grass grub, Costelytra zealandica. Journal of Microbiological Methods, 50(1), 1-8. https://doi.org/10.1016/s0167-7012(02)00004-0 

KAČÁNIOVÁ, M., KLŪGA, A., KÁNTOR, A., MEDO, J., ŽIAROVSKÁ, J., PUCHALSKI, C., TERENTJEVA, M. 2019. Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for identification of Pseudomonas species isolated from fish. Microbial Pathogenesis, 132, 313-318. https://doi.org/10.1016/j.micpath.2019.04.024

KASHINSKAYA, E. N., BELKOVA, N. L., IZVEKOVA, G. I., SIMONOV, E. P., ANDREE, K. B., GLUPOV, V. V., BATURINA, O. A., KABILOV, M. R., SOLOVYEV, M. M. 2015. A comparative study on microbiota from the intestine of Prussian carp (Carassius gibelio) and their aquatic environmental compartments, using different molecular methods. Journal of Applied Microbiology, 119(4), 948-961. https://doi.org/10.1111/jam.12904 

KELLY, D. P., MCDONALD, I. R., WOOD, A. P. 2000. Proposal for the reclassification of Thiobacillus novellus as Starkeya novella gen. nov., comb.nov. in the α-subclass of the Proteobacteria. International Journal of Systematic and Evolutionary Microbiology, 50(5), 1797-1802. https://doi.org/10.1099/00207713-50-5-1797 

KLUGA A., KACANIOVÁ M., KANTOR A., KOVALENKO K., TERENTJEVA M. 2017. Identification of microflora of freshwater fish caught in the Driksna river and pond in Latvia. 11th Baltic Conference on Food Science and Technology “Food science and technology in a changing world” FOODBALT 2017, Jelgava, Latvia, 27-28 April 2017, 164-168. https://doi:10.22616/foodbalt.2017.016

KOZIŃSKA, A. 2007. Dominant pathogenic species of mesophilic aeromonads isolated from diseased and health fish cultured in Poland. Journal of Fish Diseases, 30(5), 293-301. https://doi.org/10.1111/j.1365-2761.2007.00813.x 

LI, C., WANG, R., SU, B., LUO, Y., TERHUNE, J., BECK, B., PEATMAN, E. 2013. Evasion of mucosal defences during Aeromonas hydrophila infection of channel catfish (Ictalurus punctatus) skin. Developmental & Comparative Immunology, 39(4), 447-455. https://doi.org/10.1016/j.dci.2012.11.009 

LÜ, A., HU, X., ZHENG, L., ZHU, A., CAO, C., JIANG, J. 2011. Isolation and characterization of Citrobacter spp. from the intestine of grass carp Ctenopharyngodon idellus. Aquaculture, 313(1), 156-160. https://doi.org/10.1016/j.aquaculture.2011.01.018 

MACMILLAN, J. R., SANTUCCI, T. 1990. Seasonal trends in intestinal bacterial flora of farm-raised channel catfish. Journal of Aquatic Animal Health, 2(3), 217-222. https://doi.org/10.1577/1548-8667(1990)002<0217:stiibf>2.3.co;2 

NAVARRETE, P., MAGNE, F., MARDONES, P., RIVEROS, M., OPAZO, R., SUAU, A., POCHART, P., ROMERO J. 2010. Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss). FEMS Microbiology Ecology, 71(1), 148-156. https://doi.org/10.1111/j.1574-6941.2009.00769.x 

PIOTROWSKA-SEGET, Z., CYCOŃ, M., KOZDRÓJ, J. 2005. Metal-tolerant bacteria occurring in heavily polluted soil and mean spoil. Applied Soil Ecology, 28(3), 237-246. https://doi.org/10.1016/j.apsoil.2004.08.001 

SEKAR, V. T., SANTIAGO, T. C., VIJAYAN, K. K., ALAVANDI, S. V., RAJ, V. S., RAJAN, J. J. S., SANJUKTHA, M., KALAIMANI, N. 2008. Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus. Letters in Applied Microbiology, 46(6), 667-67. https://doi.org/10.1111/j.1472-765x.2008.02365.x 

SHAHI, H., MALLIK, S. K. 2014. Recovery of Pseudomonas koreensis from eye lesions in golden mahseer, Tor putitora (Hamilton, 1822) in Uttarakhand, India. Journal of Fish Diseases, 37(5), 497-500. https://doi.org/10.1111/jfd.12126 

SINGHAL, N., KUMAR, M., KANAUJIA. P.K., VIRDI, J.S. 2015.  MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2015.00791

SKRODENYTÉ-ARBAČIAUSKIENÉ, V. 2007. Enzymatic activity of intestinal bacteria in roach Rutilus rutilus L. Fisheries Science, 73(4), 964-966. https://doi.org/10.1111/j.1444-2906.2007.01421.x 

SKRODENYTÉ-ARBAČIAUSKIENÉ, V., SRUOGA, A., BUTKAUSKAS, D., SKRUPSKELIS, K. 2008. Phylogenetic analysis of intestinal bacteria of freshwater salmon Salmo salar and sea trout Salmo trutta trutta and diet. Fisheries Science, 74, 1307-1314.

SPANGGAARD, B., HUBER, I., NIELSEN, J., NIELSEN, T., APPEL, K. F., GRAM, L. 2000. The microflora of rainbow trout intestine: A comparison of traditional and molecular identification. Aquaculture, 182(1-2), 1-15. https://doi.org/10.1016/s0044-8486(99)00250-1 

SUGITA, H., SHIBUYA, K., HANADA, H., DEGUCHI, Y. 1997. Antibacterial abilities of intestinal microflora of the river fish. Fisheries Science, 63(3), 378-383. https://doi.org/10.2331/fishsci.63.378 

TASH, K. 2005. Rahnella aquatilis bacteremia from a suspected urinary source. Journal of Clinical Microbiology, 43(5), 2526-2528. https://doi.org/10.1128/jcm.43.5.2526-2528.2005 

TOBACK, E., DECOSTERE, A., HERMANS, K., HAESEBROUCK, F., CHIERS, K. 2007. Yersinia ruckeri infectious in salmonid fish. Journal of Fish Diseases, 30(5), 257-268. https://doi.org/10.1111/j.1365-2761.2007.00816.x 

TRYFINOPOULOU, P., TSAKALIDOU, E., NYCHAS, G. J. E. 2002. Characterization of Pseudomonas spp. associated with spoilage of gilt-head sea bream stored under various conditions. Applied and Environmental Microbiology, 68(1), 65-72. https://doi.org/10.1128/aem.68.1.65-72.2002 

VADKERTIOVÁ, R., MOLNÁROVÁ, J., VRÁNOVÁ, D., SLÁVIKOVÁ, E. 2012. Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees. Canadian Journal of Microbiology, 58(12), 1344-1352. https://doi.org/10.1139/cjm-2012-0468 

VAZ-MOREIRA, I., NUNES, O. C., MANAIA, C. M. 2012. Diversity and antibiotic resistance in Pseudomonas spp. from drinking water. Science of Total Environment, 426, 366-374. https://doi.org/10.1016/j.scitotenv.2012.03.046 

WHITE, D. C., SUTTON, S. D., RINGELBERG, D. B. 1996. The genus Sphingomonas: physiology and ecology. Current Opinion in Biotechnology, 7(3), 301-306. https://doi.org/10.1016/s0958-1669(96)80034-6 

WU, S., GAO, T., ZHENG, Y., WANG, W., CHENG, Y., WANG, G. 2010. Microbial diversity of intestinal content and mucus in yellow catfish (Pelteobagrus fulvidraco). Aquaculture, 303(1-4), 1-7. https://doi.org/10.1016/j.aquaculture.2009.12.025