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Article

Molecular Identification of Bacteria Isolated from Marketed Sparus aurata and Penaeus indicus Sea Products: Antibiotic Resistance Profiling and Evaluation of Biofilm Formation

by
Mohammad A. Abdulhakeem
1,
Mousa Alreshidi
1,2,*,
Fevzi Bardakci
1,
Walid Sabri Hamadou
1,
Vincenzo De Feo
3,
Emira Noumi
1,4,* and
Mejdi Snoussi
1,4
1
Department of Biology, College of Science, University of Ha’il, Ha’il P.O. Box 2440, Saudi Arabia
2
Molecular Diagnostics and Personalized Therapeutics Unit, University of Hail, Hail P.O. Box 2440, Saudi Arabia
3
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, Fisciano, 84084 Salerno, Italy
4
Laboratory of Genetics, Biodiversity and Valorization of Bio-Resources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Avenue Tahar Haddad, BP74, Monastir 5000, Tunisia
*
Authors to whom correspondence should be addressed.
Life 2023, 13(2), 548; https://doi.org/10.3390/life13020548
Submission received: 2 January 2023 / Revised: 6 February 2023 / Accepted: 8 February 2023 / Published: 16 February 2023
(This article belongs to the Special Issue Antibiotic Resistance in Biofilm)
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Abstract

:
Background: Marketed fish and shellfish are a source of multidrug-resistant and biofilm-forming foodborne pathogenic microorganisms. Methods: Bacteria isolated from Sparus aurata and Penaeus indicus collected from a local market in Hail region (Saudi Arabia) were isolated on selective and chromogenic media and identified by using 16S RNA sequencing technique. The exoenzyme production and the antibiotic susceptibility patterns of all identified bacteria were also tested. All identified bacteria were tested for their ability to form biofilm by using both qualitative and quantitative assays. Results: Using 16S RNA sequencing method, eight genera were identified dominated by Vibrio (42.85%), Aeromonas (23.80%), and Photobacterium (9.52%). The dominant species were V. natrigens (23.8%) and A. veronii (23.80%). All the identified strains were able to produce several exoenzymes (amylases, gelatinase, haemolysins, lecithinase, DNase, lipase, and caseinase). All tested bacteria were multidrug-resistant with a high value of the multiple antibiotic index (MARI). The antibiotic resistance index (ARI) was about 0.542 for Vibrio spp. and 0.553 for Aeromonas spp. On Congo red agar, six morphotypes were obtained, and 33.33% were slime-positive bacteria. Almost all tested microorganisms were able to form a biofilm on glass tube. Using the crystal violet technique, the tested bacteria were able to form a biofilm on glass, plastic, and polystyrene abiotic surfaces with different magnitude. Conclusions: Our findings suggest that marketed S. aurata and P. indicus harbor various bacteria with human interest that are able to produce several related-virulence factors.

1. Introduction

Seafood has great nutritional benefits and economic importance; thus, the bacterial species present in seafood must be identified and studied to determine the best health practices to prevent seafood-borne illnesses [1]. Fish is the food category mainly associated with foodborne outbreaks, accounting for approximately 6–8% of the total food-borne diseases. This prevalence is greater than the incidence of food illness cases from chicken and beef [2]. Both pathogenic and harmful bacteria can be introduced into seafood products during the manufacturing process and in the supply chain [3,4]. Shellfish are considered as a major source of seafood-borne pathogens in humans, as they are usually consumed undercooked or raw. Water warming due to climate change has recently become an issue as it would elevate the microbial population, including Vibrio species in particular foodborne strains and other pathogenic bacteria that ultimately end up in seafood environments, inducing more seafood-borne diseases as a result of the intake of contaminated seafood. Fish and other seafood are a source of various microorganisms with human health interest including Gram-negative bacteria (Pseudomonas, Shewanella, Psychrobacter, Pseudoalteromonas, Moraxella, Acinetobacter, Flavobacterium, Vibrio, Photobacterium, and Aeromonas) and Gram-positive bacteria (lactic acid bacteria (LAB), Micrococcus, Corynebacterium, Vagococcus, Bacillus, and Clostridium) [5]. Pathogenic bacteria in seafood can be transmitted to humans during food intake, inducing serious health issues, including cellulitis and septicemia. Pathogens can also enter the bloodstream through wounds or open cuts while handling infected seafood or swimming, causing necrotizing fasciitis and fatal septicemia in susceptible individuals [6,7]. The high incidence of seafood poisoning indicates substantial challenges in controlling pathogenic microbes that induce food-borne illnesses [8,9]. The microbial diversity in fish is highly related to the conditions in which they live and remain after harvesting. Microbial diversity can be determined by a wide range of parameters, including location, origin, water type (e.g., brackish or freshwater), and catching and handling processes [10]. Understanding the prevalence, ecology, concentration, and dynamics of pathogenic and spoilage microorganisms in seafood would contribute to developing effective preservative mechanisms. Antibiotics are widely used in aquaculture for prophylactic and therapeutic purposes. However, the misapplication of antibiotics has significantly contributed to the increase in antibiotic-resistant microbes (ARMs) and resistance genes in aquaculture farms and neighboring coastal settings [11]. In addition, the excessive use of antimicrobials could lead to widespread multidrug-resistant microorganisms in fish, shellfish, and their surrounding water [12,13,14,15,16].
The prevalence of ARMs has become a worldwide issue in all food plants, not only in seafood. ARMs have become a major concern for public health, and many isolates from seafood have demonstrated a higher degree of resistance against a wide range of antibiotics [17].
Hence, the main objective of this study was to identify the main bacteria in Sparus aurata and Penaeus indicus, which are highly consumed in the Ha’il region. Further importance was given to the determinants of antibiotic resistance and biofilm formation in the identified isolates. The current study also investigated the ability of these isolates to produce different enzymes. It was hypothesized that the isolated bacteria would have different degrees of antibiotic resistance and biofilm formation, as well as different exoenzyme profiles.

2. Materials and Methods

2.1. Sampling Material and Bacterial Isolation

Bacteria were isolated from gilthead sea bream (Sparus aurata L.) and shellfish (Penaeus indicus H. Milne-Edwards). These samples were obtained from a local market in Hail region-Saudi on 25 February 2022. Fish with red spots on their skin were targeted, as this is an indication of microbial infection. Upon arrival, gilthead sea bream and prawns were immediately washed using sterile seawater, gutted, headed, and shucked with a sterile knife. Twenty-five grams from prawn abdomen meat, and the intestines, gills, and muscle meat from S. aurata were enriched in 225 mL of alkaline peptone water supplemented with 1% NaCl [18]. The inoculated broth media was incubated overnight at 37 °C. After incubation, a loopful from each enrichment culture was steaked onto thiosulfate–citrate–bile salt–sucrose agar (TCBS) (Agar; Sigma Aldrich, Darmstadt, Germany) and onto Vibrio ChromoSelect agar (Sigma Aldrich, Germany), before incubating for 18 to 24 h at 37 °C.

2.2. Bacterial Identification and Phylogenetic Analyzes

Twenty-one bacterial isolates were selected from both selective and chromogenic media (seven isolates from P. indicus abdomen muscles, six isolates from S. aurata intestines, four isolates from S. aurata muscle meat, and four isolates from S. aurata gills). The DNA extraction was conducted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The polymerase chain reaction (PCR) was performed using Applied BiosystemsTM (Waltham, MA, USA) apparatus, with the following PCR conditions: initial denaturing step for 5 min, 30 cycles of amplification (30 s denaturation at 95 °C, 30 s annealing step at 52 °C, and 30 s extension at 72 °C), and a final extension step at 72 °C for 5 min. After PCR, the obtained products of the 27F-907R regions were electrophoresed using 2% agarose gel (110 V, 150 mA, 45 min). It was observed that the size of the products was between 1200–1400 bp. Then, the cleanup phase was started using ExoSAP-IT Express PCR Cleanup Reagents. For the experiment, 10 µL of each PCR product was mixed with 4 µL of the cleanup reagent.
Molecular identification of isolated bacteria was achieved by sequencing the 16S rDNA gene using the universal primer 27F (5′–AGAGTTTGATCMTGGCTCAG–3′) and the reverse primer 907R (5′–CCGTCAATTCCTTTRAGTTT–3′) previously described by Muyzer et al. [19]. After the DNA sequencing reaction was completed, the sequencing products were purified using the gel filtration method with Sephadex. After the purification process, the DNA sequencing process was started. This was performed on the ABI 3130XL device using the capillary electrophoresis method.
Sequence alignment was performed using BioEdit version 7.1.3.0 [20], and a total of 859 bp were successfully aligned in the final dataset consisting of 31 nucleotide sequences, including Salmonella enterica as the outgroup. The 16S rDNA sequences were subjected to a BLAST search to determine the sequence homology with the sequences previously deposited in the NCBI to identify isolated bacterial species and strains. The sequences with the highest homology belonging to Vagococcus fluvialis, Staphylococcus aureus, Staphylococcus epidermidis, Shewanella indica, Photobacterium damselae, Morganella morganii, Bacillus cereus, Aeromonas veronii, and Vibrio harveyi were added to our dataset to determine the relation of our isolates with them. A phylogenetic tree was generated by the neighbor-joining method [21] with a bootstrap test (1000 replicates) [22]. The Kimura two-parameter method [23], the best-fitting model for our sequence dataset, was used to compute the pairwise evolutionary distances among the sequences, with the gaps removed by the pairwise deletion option implemented in MEGA 11 software [24].

2.3. Exoenzyme Production

The identified isolates were tested for their abilities to produce several exoenzymes including DNAase, lipase, amylase, caseinase, and lecithinase, according to the protocol described by Hörmansdorfer et al. [25] and Snoussi et al. [26]. For the experiment, PBS agar medium was supplemented with Tween-80 (lipase activity), starch (amylase activity), skim milk powder (caseinase activity), and egg yolk (lecithinase production). Petri dishes were incubated for 72 h at 37 °C, and the formation of a clear zone around the inoculated spots was considered a positive test. In addition, hemolysin production was tested on human blood agar supplemented with 5% human blood (Oxoid Ltd., Basingstoke, UK) [26].

2.4. Antibiotic Susceptibility Test

Susceptibility to several antimicrobial agents was determined using the disc diffusion assay on Mueller–Hinton agar/1% NaCl [14,27]. The following antibiotics (Oxoid, UK) were tested against all identified bacteria: amikacin (AK, 30 μg), ampicillin (AMP, 10 μg), gentamicin (GEN, 10 μg), tetracycline (TET, 10 μg), ertapenem (ETP, 10 μg), fosfomycin (FOS, 200 μg), norfloxacine (NOR, 10 μg), linezolid (LZD, 30 μg), nitrofurantoin (F, 100 μg), ciprofloxacin (CIP, 5 μg), nalidixic acid (NA, 30 μg), moxifloxacin (MXF, 30 μg), meropenem (MEM, 10 μg), ticarcillin (TIC, 75 μg), piperacillin + tazobactam (PPT, 75/10 μg), cefotaxime (CTX, 30 μg), tigecycline (TGC, 15 μg), pristinamycin (PTN, 15 μg), rifampicin (RAM, 30 μg), erythromycin (E, 15 μg), chloramphenicol (C, 30 μg), amoxicillin + clavulanic acid (AUG, 30 μg), temocillin (TMO, 30 μg), tobramycin (TOB, 10 μg), sulphamethoxazle + trimethoprim (SXT, 25 μg), ceftazidime (CZD, 30 μg), ceftaroline (CPN, 5 μg), colistin (CST 50 μg), netilmicin (NET, 30 μg), and teicoplanin (TEC, 30 μg). After incubation at 37 °C for 18 to 24 h, the diameter of the inhibition zone was measured using a 1 mm flat rule. The antibiotic susceptibility profile of the isolate was interpreted as sensitive, intermediate, and resistant according to the Clinical and Laboratory Standards Institute (CLSI) M45 and (CLSI) M100 guidelines Institute [28,29]. Two mathematic indices were used to interpret the results obtained: (i) the antibiotic resistance index (ARI) of each bacterial population [30], and (ii) the multiple antibiotic resistances (MAR) index of the isolates [31].

2.5. Adhesion Properties and Biofilm Formation Screening

2.5.1. Exopolysaccharide (Slime) Production

The ability of all identified bacteria to secrete an exopolysaccharide layer (slime production) was tested using the same protocol previously described by Snoussi et al. [32] adapted to Vibrio species. Colonies obtained on Congo red agar were interpreted as slime producers (pigmented colonies), while unpigmented colonies were interpreted as slime nonproducers [33].

2.5.2. Wolfe Test

The ability of the identified bacteria to adhere to the glass surface was tested using the same protocol described by Wolfe et al. [34]. For the experiment, a 10 mL glass tube (0.5 cm in diameter) containing seawater broth (5 g of Bactotryptone, 3 g of yeast extract, and 3 mL of glycerol in 700 mL of seawater and 300 mL of purified water) was used to grow overnight all bacteria at 37 °C. Afterward, 100 μL of this pre-enriched culture were added to inoculate new tubes containing the same medium and incubated at 37 °C for 10 h without shaking. Following a 15 min staining period with 1% (w/v) crystal violet, all glass tubes were cleaned with distilled water before being used for further testing. Glass-biofilm positives were bacteria that produced a purple pellicule on the cultures’ air surface.

2.5.3. Biofilm Formation on Polystyrene Microtiter Plates

The capacity of bacteria to create a biofilm on 96-well polystyrene microtiter plates was estimated using the Toledo-Arana et al. protocol [35]. Brain Infusion Broth (BHI/0.25% (w/v) glucose) medium was used for the pre-enrichment of all bacterial strains. After overnight culture of the tested bacterial in a microtiter plate for 24 h at 37 °C, adherent bacteria were stained using crystal violet (1%) for 15 min, and then solubilized with ethanol–acetone (80:20 v/v). The optical density (OD595nm) was measured spectrophotometrically. Bacteria were interpreted as (−) non-biofilm forming OD595 ≤ 1, (+) weak biofilm forming 1 < OD595 ≤ 2, (++) medium biofilm forming 2 < OD595 ≤ 3, or (+++) strong biofilm forming OD595 > 3 [33]. Each essay was performed three times.

2.5.4. Biofilm Formation on Glass and Plastic Surfaces

Glass material (circular 12 mm diameter cover glasses) and a plastic surface (12 mm diameter) were used for the quantitative estimation of biofilm-forming capacities of all identified strains from S. aurata and P. indicus inserted into the bottom of 24-well (15 mm diameter each well) microtiter plates and filled with 2 mL of each bacterial suspension (109 UFC/mL in PBS) for 24 h at 37 °C. The experiments were carried out in triplicate and three times. The same procedure described by Henriques et al. [36] was followed using 600 μL of crystal violet for 5 min to stain the biofilm-forming bacteria fixed on the abiotic surfaces selected. The pieces were gently washed in water and dried before being immersed in 1 mL of 33% (w/v) acetic acid to release and dissolve the stain. Using a microtiter plate reader, the OD of the resulting solution was measured at 570 nm (Bio-Tek, Model Synergy HT, city). Results were interpreted using the scheme proposed by Stepanović et al. [37], where bacteria were interpreted as nonadherent (0) OD ≤ ODc, weakly adherent (+) ODc < OD ≤ 2 × ODc, moderately adherent (++) 2 × ODc < OD ≤ 4 × ODc, or strongly adherent (+++): 4 × ODc < OD.

2.6. Statistical Analysis

All experiments were performed in triplicate, and the average and standard deviation were calculated using the SPSS 25.0 statistical package for Windows.

3. Results

3.1. Morphological Characterization and 16SRNA Identification of Bacterial Isolates

The analysis of different samples from P. indicus on TCBS agar medium revealed the characterization of two morphotypes: yellow colonies of 1 to 2 mm in diameter and green-yellow colonies with a diameter of about 2–3 mm, respectively. In addition, on Vibrio ChromoSelect agar, four different morphotypes based on color and size were observed (Table 1). Similarly, 14 morphotypes were obtained from S. aurata after being cultured on TCBS and Vibrio ChromoSelect agar medium. The dominant color on TCBS agar plates was yellow (diameter between 1 and 7 mm). However, on Vibrio ChromoSelect agar, nine isolates with various ranges of colony shapes, sizes, and colors were seen, including blue, turquoise, purple, pink, light green with a green center, and colorless colonies (Table 1).
Twenty-one colonies were subsequently analyzed using molecular techniques (16S RNA) and bioinformatics (BioEdit software and National Center for Biotechnology Information (NCBI)) to certain the identity of these isolates. The results demonstrated that the main bacteria identified in both samples belonged to the Vibrio genus with four different species, including V. natriegens, V. harveyi, V. alginolyticus, and V. hyugaensis. The second most dominant species was Aeromonas veronii. The phylogenetic tree (Figure 1) shows the genetic relationship of the 21 identified bacterial strains, isolated from various organs of S. aurata and shrimps (P. indicus) using the NJ method.

3.2. Exoenzyme Production

The bacteria identified in this study were investigated for their capability to produce several hydrolytic enzymes. The results demonstrated that all tested bacteria were able to produce amylase (100%), but other enzymes were only induced by some bacteria. The percentage of the total bacteria secreted by each enzyme was as follows: lipase (80.95%), DNase (71.42%), caseinase (66.66%), lecithinase (57.14), hemolysins (52.38), and gelatinase (47.61%) (Table 2). Some bacteria were able to produce all enzymes tested, such as V. harveyi (P9), V. alginolyticus (P2), A. veronii (SA15), V. fluvialis (SA21). It was observed that some bacteria with the same identify had different exoenzyme profiles.

3.3. Antibiotic Susceptibility Test

The results of the antibiotic susceptibility test showed that some of the tested bacteria were resistant to all antibiotics used with the exception of norfloxacin and trimethoprim–sulfamethoxazole which were effective against all Gram-negative bacteria (Supplementary Material Table S1). The analysis indicated that all Gram-negative bacteria were completely resistant to tigecycline, ceftaroline, meropenem, and ticarcillin and highly resistant to amikacin (94.44%), ampicillin (4.44%), amoxicillin + clavulanic acid (88.88%), gentamicin (83.33%), and moxifloxacin (83.33%) (Figure 2). The lowest percentage of resistance was recorded for the following antibiotics: norfloxacin (0%), trimethoprim–sulfamethoxazole (0%), tobramycin (5.55%), netilmicin (5.55%), chloramphenicol (5.55%), and nalidixic acid (16.66%).
In addition, it is worth noting that Vibrio spp. (n = 9) were particularly completely resistant to ceftaroline, tigecycline, ticarcillin, colistin, and meropenem (Figure 3). On the other hand, these bacteria were found to be sensitive or dose-dependently resistant (intermediate) to netilmicin, norfloxacin, chloramphenicol, and trimethoprim–sulfamethoxazole. Similarly, the Aeromonas spp. (n = 5) strains were completely resistant to amikacin, moxifloxacin, ceftaroline, tigecycline, amoxicillin–clavulanic acid, ampicillin, ticarcillin, and meropenem. The lowest percentage of resistance was recorded with three antibiotics (tobramycin, norfloxacin, and trimethoprim–sulfamethoxazole) (Figure 3).
Overall, the tested bacteria could be considered as multidrug-resistant microorganisms, as all isolates were resistant to three or more antibiotics from different classes (Supplementary Tables S1 and S2). In fact, the multiple antibiotic resistance index (MARI) for Vibrio spp. (n = 9) ranged from 0.384 (V. harveyi P9) to 0.653 (V. alginolyticus P2). Regarding the Aeromonas spp. strains (n = 5), the MARI ranged from 0.461 (A. veronii SA17) to 0.692 (A. veronii SA25). The MARI ranged from 0.423 (M. morganii P5) to 0.653 (S. indica P13) for the other Gram-negative identified bacteria. Similarly, for Gram-positive bacteria, the MARI was about 0.5 for B. cereus SA9, 0.333 for S. epidermidis SA7, and 0.277 for V. fluvialis SA21. In addition, our results revealed that the calculated ARI varied from 0.542 for all Vibrio strains (n = 9) to 0.553 for all Aeromonas strains (n = 5). Taken together, the ARI for the 18 Gram-negative bacteria was about 0.544, while the same index was lower (ARI = 0.462) for the Gram-positive bacteria tested (Table 3).

3.4. Slime Production on CRA Plates and Glass Tubes (Wolfe Test)

The phenotypic production of slime was assessed by culturing the bacteria on Congo Red Agar plates. Pigmented colonies were considered as normal slime-producing bacteria, whereas colorless colonies were classified as non-slime-producing. Among the tested isolates, six out of 21 (28.57%) were able to induce slime, indicated by black colonies, and the remaining 15 bacteria were non-slime-producing characterized by red, orange, Bordeaux, white with a red center, or black-gray morphotypes (Figure 4).
All tasted bacteria were able to adhere to the glass, giving a purple pellicule on the air surface of the glass tube, except for Photobacterium damselae. The intensity of the color of the crust formed ranged from intense to moderate. Moreover, 10 bacteria out of 21 tested were strongly adhesive to glass surface (Figure 5). All these data are summarized in Table 4.

3.5. Quantitative Estimation of Biofilm Formation by Tasted Bacteria on Abiotic Surfaces

On a polystyrene 96-well microtiter plate (U-bottom), five bacteria (V. natrigens SA11, V. alginolyticus P2, V. hyugaensis P12, P. piscicida SA3, and B. cereus SA9) out of 21 were medium biofilm forming with an optical density of about 2 < OD595 ≤ 3. In addition, 12 bacteria weakly adhered to polystyrene and formed a weak biofilm (1 < OD595≤ 2) on the polystyrene surface (96-well plate). Interestingly, all tested bacteria were able to adhere to glass and plastic surfaces to different degrees, with the exception of only two strains (V. natrigens SA11 and V. hyugaensis P12) on glass and three strains on plastic, namely, V. hyugaensis P12, A. veronii SA15, and A. veronii SA25, regardless of their origin (S. aurata or P. indicus samples). In fact, two bacteria isolated from P. indicus (V. natrigens P14 and S. indica P13) and one from S. aurata (V. natrigens SA11) adhered strongly to the plastic surface, whereas no bacteria were strongly biofilm forming on glass material. Overall, 9/21 (42.85%) formed a biofilm on polystyrene, in contrast to 19/21 (90.47%) on glass and 18/21 (85.71%) on the plastic surface, to different degrees ranging from weak to strong. Interestingly, six bacteria (V. harveyi P9, V. alginolyticus P12, A. veronii SA1, A. veronii SA31, P. piscicida SA3, and B. cereus SA9) were able to adhere to all tested surfaces to different degrees. Lastly, in particular, B. cereus (SA9) isolated from the intestines of S. aurata was moderately able to adhere to all tested surfaces (polystyrene, glass, and plastic) (Table 5).

4. Discussion

The current study identified bacteria in fish and shrimp (S. aurata and P. indicus) and subsequently investigated their ability to produce different arrays of extracellular enzymes, their biofilm formation ability, and their antibiotic susceptibility. The results identified eight genera of bacteria in the analyzed samples. The abundant bacterial species found were from the Vibrio and Aeromonas genera, accounting for nine and five species, respectively. These findings consistent with previous publications that showed that Vibrio, Aeromonas and Photobacterium genera were frequently identified in marketed sea food products from around the world [38,39,40,41], as well as in Saudi Arabia [42,43,44,45,46,47,48,49,50]. In fact, Al-Sunaiher and colleagues [42] reported the isolation 62 Vibrio spp. strains belonging mainly to V. hollisae (54.5%), V. fluvialis (20.5%), V. damselae (12.6%), V. alginolyticus (6.8%), and V. vulnificus (4.5%) from Oreochromis niloticus L., O. spilurus L., Mugil cephalus L., Dicentrarchus labrax L., Siganus rivulatus L., and Carus gariepinus L. In 2016, Elhadi and colleagues [44] reported the prevalence of E. coli (18.6%), Enterococci (14.4%), Pseudomonas (14%), and Salmonella (16.8%) in imported frozen fish (Pangasius pangasius, Cirrhinus mrigala, Oreochromis niloticus, Cyprinus carpio, Labeo rohita, Chanos chanos, and Rastrelliger brachysoma) from a local market in Eastern province of Saudi Arabia. Similarly, using the 16S rDNA technique, Alikunhi and colleagues [43] identified different bacteria from 13 edible fish species from Jeddah province, namely, P. stutzeri, V. harveyi, Aeromonas sp., A. salmonicida, Rahnella aquatilis, V. damselae, Hafnia sp., Pseudoalteromonas sp., and Psychrobacter faecalis.
More recently, Beyari and colleagues [49] studied the bacterial diversity in some marketed fish retails from Jeddah province and reported the identification of 17 different bacterial genera (dominated by Aeromonas, Pseudomonas, Psychrobacter, and Alcaligens). The same authors reported the identification of 32 different species including some human pathogenic ones such as R. aquatilis, Proteus vulgaris, Klebsiella quasipneumoniae, Yersinia enterocolitica, P. lundensis, P. oryzihabitans, Psychrobacter phenylpyruvicus, P. sanguinis, Alcaligenes faecalis, and P. putida.
The presence of pathogenic bacteria in fish and other seafood is thought mainly to result from the growth conditions, harvesting, and preservation processes that support the spread of microorganisms, particularly pathogens. Therefore, it has been found that cautious processing methods could significantly reduce microbes in fish and other seafood [51]. The major isolated bacteria found in the study were Gram-negative bacteria. This result is similar to previous publications [52]. The main bacteria identified in the current study were Vibrio species, accounting for 43% of the total bacteria identified. Vibrio species, including V. harveyi, V. alginolyticus, V. natriegens, and V. hyugaensis are associated with many human and fish diseases. The next most abundant genus was Aeromonas veronii in both fish and shrimp; this bacterium, along with other Aeromonas species, has been linked to diarrhea cases in children [53], where approximately 8% of acute enteric infections are induced by Aeromonas species [54]. It was found that most Aeromonas species could be isolated from different environments, including rivers, meat, and fish, as well as from patients suffering from diarrhea [55,56,57].
Thus, Aeromonas species are considered to be primary pathogens in aquaculture that can grow at refrigerator temperature and, hence, can be a major source of food contamination, especially where is a probability of cross-contamination with prepared-to-consume food products [58]. Recently, many fish infections have been initiated by Aeromonas species [59,60,61,62]. Other bacterial genera detected in this study include Shewanella, Photobacterium, Vagococcus, Staphylococcus, and Bacillus. Bacteria can live comfortably in aquatic settings; thus, bacterial infection has become the main barrier to the success of aquaculture farms [63].
This investigation also looked at the ability of the identified bacteria to produce extracellular enzymes, which have been recognized as an indicator of health risk in microbes isolated from different sources, including clinical, food, and environmental samples [64,65]. It was found that the bacterial isolates could yield at least two exoenzymes, including amylase, the only enzyme was produced by all isolates. These results indicated that all isolated bacteria produced a variety of extracellular enzymes, but each isolate had a distinct pattern of hydrolytic enzymes. Enzymes produced by bacteria could potentially modulate the bacterial virulence and pathogenicity, breaking down proteins and making them available for proliferation [66,67]. The secretion of some enzymes and toxins has been found to be responsible for food spoilage and can make the bacteria more resistant to antibiotic agents, leading to therapeutic issues [68]. Amylase was the only enzyme produced by all isolated bacteria, which may indicate the capability of all isolates to use this enzyme to hydrolyze starch [69]. Almost 50% of Vibrio isolates in the current study expressed all exoenzymes tested, with complete production of lipase and amylase. In a similar manner, several extracellular enzymes were produced by Vibrio [33,68]. Approximately half of the bacterial isolates were capable of producing hemolysin and gelatinase; these enzymes are recognized as virulence factors as both are associated with bacterial pathogenicity [70]. In addition, 80% of the isolates had lipolytic activity, which is associated with the acquisition of nutrients by degrading host lipids. More than 66% of the isolates had DNase and caseinase activities. DNase has a function as an endonuclease and partially plays a role in DNA hydrolysis, whereas caseinase is associated with bacterial pathogenicity. Several exoenzymes have been detected in Vibrio bacteria from different sources, including fish, shrimp, and shark [71,72].
The analysis of antibiotic resistance indicated that all Gram-negative bacteria were completely resistant to tigecycline, ceftaroline, meropenem, and ticarcillin. Moreover, Vibrio spp. were particularly completely resistant to ceftaroline, tigecycline, ticarcillin, colistin, and meropenem, whereas Aeromonas spp. strains were completely resistant to amikacin, moxifloxacin, ceftaroline, tigecycline, amoxicillin–clavulanic acid, ampicillin, ticarcillin, and meropenem. The antibiotic resistant indices (ARIs) for Vibrio and Aeromonas species were approximately 0.542 and 0.553, respectively. Examination of antibiotic resistance in bacteria isolated from fish and shellfish is essential in order to estimate the level of changes in water ecosystems as a result of human activities [73]. The analysis revealed that the ARI of all the Gram-negative bacteria was 0.544, whereas that of Gram-positive bacteria was 0.305. It has been proposed that bacteria with ARI indices greater than two could be a potential source of high risk where chemicals, including antibiotics, are in high use [74]. These two indices (MARI and ARI) were previously used to study the distribution of multidrug-resistant microorganisms associated with fish and shellfish products [38]. In fact, Al-Sunaiher and colleagues [42] found that V. vulnificus, V. damselae, V. fluvialis, V. hollisae, and V. alginolyticus species were resistant to several antibiotics tested with high percentages: amoxycillin (66.7–100%), oxytetracycline (33.3–100%), ampicillin (33.3–100%), penicillin (79–100%), chloramphenicol (0–37.5%), sulfonamide (70–100%), cloistin (0–66.7%), tetracycline (6.7–100%), lincomycin (62.5–100%), trimethoprim (20–100%), nalidixic acid (0–63.3%), nitrofurantoin (0–100%), and oxolinic acid (0–100%). Interestingly, the same team reported that the isolated V. vulnificus strains were resistant (100%) to trimethoprim, tetracycline, sulfamethoxazole, penicillin, oxytetracycline, ampicillin, amoxicillin, and lincomycin. Al-Ghanayem and colleagues [50] reported the identification of Aeromonas spp., E. coli, Enterobacter spp., Proteus spp., Enterococcus spp., and Streptococcus spp. strains from the local fish market at Shaqra (Riyadh province, Saudi Arabia) highly resistant to amoxicillin, bacitracin, chloramphenicol, ciprofloxacin, erythromycin, gentamycin, and tetracycline with a multidrug resistance index ranging from 0.33 for Streptococcus species to 0.44 for E.coli strains.
The result of phenotypic slime production revealed that 28% of the total isolates were positive slime producers. These isolates belong to the Vibrio and Anemones genera. Slime production is measured as an important virulence factor in some pathogenic bacteria, including Vibrio and Aeromonas species, and it could be an indicator of a high-risk commination [75]. Slime is used by bacteria as a protective mechanism against external environments; thus, microbes coated with slime are more resistant to antibiotics and other stressors. Slime molecules are considered to be involved in biofilm formation; indeed, they play a significant role in the initial stages of biofilm development [76,77]. Previous studies have reported the characterization of several morphotypes formed by V. alginolyticus isolated from fish (S. aurata, D. labrax) on Congo red agar, and most of them were slime producers with black colonies [32,33]. Similarly, Snoussi and colleagues [16] reported the identification of A. hydrophila, Staphylococcus spp., V. alginolyticus, Enterobactercloacae, K. ornithinolytica, K. oxytoca, and Serratia odorifera from seabass, seabream, roseshrimp, and blue mussel. These strains produced five morphotypes based on the colorimetric scale on the tested medium (Bordeaux, red with dark center, pink with red center, pink, and red colonies).
The capacity of the isolates to form biofilms on different materials, including polystyrene, glass, and plastic, was investigated. The analysis indicated that 42.8% of the isolates formed biofilms on polystyrene, in contrast to 90.4% on glass and 85.7 on plastic, to varying degrees ranging from weak to strong. Biofilm development seemed to be affected by surface properties; the use of polystyrene materials is highly recommended to avoid biofilm formation [78]. Some isolates, including V. alginolyticus, A. veronii, P. piscicida, and B. cereus, tended to form biofilms on all tested surfaces. The results of this study are similar to others demonstrating that Aeromonas and Vibrio species from fish and shellfish and their surrounding water were able to form biofilms on different biotic and abiotic surfaces to different degrees [32,33,79]. Bacteria that develop biofilms are greatly resistant to changing environments, including antibiotics and detergents [80,81]. Thus, microbial biofilm development is a topic of important interest in many fields, including food and medical industries, as it is a significant contributor to bacterial virulent, which can lead to serious infections that are difficult to treat [82,83]. The precipitation of mineral and food residues in food manufacturing could positively affect the development of biofilms [84].

5. Conclusions

This investigation provided clear evidence that both fish and shrimp collected from local markets, having been initially harvested from an aquaculture farm, had a diversity of bacterial genera. The outcomes of this study revealed that the main bacterial genera identified were Vibrio and Aeromonas. Antimicrobial resistance was also demonstrated in all bacterial isolates, and high multidrug resistance indices were obtained in most of the tested isolates. The majority of isolates were biofilm producers, suggesting a significant threat from these isolates in the food industry. Therefore, control and prevention of microbial contamination must be taken into consideration in order to obtain healthy and uncontaminated food, particularly seafood.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/life13020548/s1. Table S1. Antibiotic susceptibility of 18 Gram negative bacteria to 26 antibiotics and MARI calculation; Table S2. Antibiotic susceptibility of V. fluvialis, B. cereus and S. epidermidis to 18 antibiotics and MARI calculation.

Author Contributions

Conceptualization, M.A.A., M.S., V.D.F. and M.A.; methodology, M.A.A., M.S., E.N. and M.A.; software, M.A.A., W.S.H. and F.B.; validation, M.A.A., M.S. and M.A.; formal analysis, M.A.A., M.S. and M.A.; investigation, M.A.A.; data curation, M.A.A., M.S. and M.A.; writing—original draft preparation, M.A.A., M.S., E.N., F.B. and M.A.; writing—review and editing, M.A.A., M.S., E.N., F.B. and M.A.; supervision, M.S. and M.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been funded by Scientific Research Deanship at the University of Ha’il, Ha il, Saudi Arabia through project number GR-22 028.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are presented in the manuscript.

Acknowledgments

This research has been funded by Scientific Research Deanship at the University of Ha’il-Saudi Arabia through project number GR-22 028.

Conflicts of Interest

The authors declare no conflict of interest.

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Life 13 00548 g001 550
Figure 1. Phylogenetic tree of the 21 identified bacteria from P. indica and S. aurata based on their 16S RNA gene sequences obtained using the UPGMA method.
Figure 1. Phylogenetic tree of the 21 identified bacteria from P. indica and S. aurata based on their 16S RNA gene sequences obtained using the UPGMA method.
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Figure 2. Percentage resistance (%) of 18 Gram-negative bacteria isolated from S. aurata and P. indicus to 26 antibiotics.
Figure 2. Percentage resistance (%) of 18 Gram-negative bacteria isolated from S. aurata and P. indicus to 26 antibiotics.
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Figure 3. Percentage resistance of Vibrio spp. (A) and Aeromonas spp. (B) strains to the 26 antibiotics tested in this study.
Figure 3. Percentage resistance of Vibrio spp. (A) and Aeromonas spp. (B) strains to the 26 antibiotics tested in this study.
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Life 13 00548 g004 550
Figure 4. Different morphotypes described on Congo red agar plates: (A) white with red center; (B) orange; (C) red; (D) black; (E) Bordeaux; (F) black-gray.
Figure 4. Different morphotypes described on Congo red agar plates: (A) white with red center; (B) orange; (C) red; (D) black; (E) Bordeaux; (F) black-gray.
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Figure 5. Pellicule formation on the surface of the tested glass tube stained with 1% crystal violet. (++): large pellicule formation; (+) weak pellicule formation; (−) no pellicule formation.
Figure 5. Pellicule formation on the surface of the tested glass tube stained with 1% crystal violet. (++): large pellicule formation; (+) weak pellicule formation; (−) no pellicule formation.
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Table
Table 1. The 16S RNA identification results and morphological characteristics of the 21 bacterial isolates from P. indicus and S. aurata obtained from TCBS and Vibrio ChromoSelect agar medium.
Table 1. The 16S RNA identification results and morphological characteristics of the 21 bacterial isolates from P. indicus and S. aurata obtained from TCBS and Vibrio ChromoSelect agar medium.
AgarSite of IsolationColony ColorCodeBacteria NameAccession
TCBS agarP. indicus abdomen musclesYellowP12Vibrio hyugaensisOP703739.1
P. indicus abdomen musclesGreen yellowP13Shewanella indicaOP704022.1
P. indicus abdomen musclesYellowP14Vibrio natriegensOP703736.1
Vibrio ChromoSelect agarP. indicus abdomen musclesBlueP1Vibrio natriegensOP703737.1
P. indicus abdomen musclesTurquoiseP2Vibrio alginolyticusOP703632.1
P. indicus abdomen musclesColorlessP5Morganella morganiiOP704015.1
P. indicus abdomen musclesPurpleP9Vibrio harveyiOP704026.1
TCBS agarS. aurata intestinesYellowSA1Aeromonas veroniiOP704025.1
S. aurata intestinesYellowSA3Photobacterium. PiscicidaOP704011.1
S. aurata musclesYellowSA17Aeromonas veroniiOP704024.1
S. aurata musclesBlue greenSA21Vagococcus fluvialisOP704018.1
S. aurata gillsYellowSA27Vibrio natriegensOP703612.1
Vibrio ChromoSelect agarS. aurata intestinesTurquoiseSA5Photobacterium damselaeOP704023.1
S. aurata intestinesLight green with a green centerSA7Staphylococcus epidermidisOP704017.1
S. aurata intestinesColorlessSA9Bacillus cereusOP704016.1
S. aurata gillsLight green with a green centerSA11Vibrio natriegensOP704027.1
S. aurata gillsColorlessSA13Vibrio natriegensOP704012.1
S. aurata gillsPinkSA15Aeromonas veroniiOP703806.1
S. aurata intestinesGreenSA25Aeromonas veroniiOP704013.1
S. aurata musclesBlueSA26Vibrio harveyiOP703738.1
S. aurata musclesColorlessSA31Aeromonas veroniiOP704014.1
Table
Table 2. Exoenzyme production by the identified bacterial strains.
Table 2. Exoenzyme production by the identified bacterial strains.
CodeStrainsDNaseLipaseLecithinaseCaseinaseHemolysisAmylaseGelatinase
P1V.natriegens++++−+−
P14V.natriegens++−−−+−
SA11V.natriegens++−−−+−
SA13V.natriegens−+++−++
SA27V.natriegens++−−−+−
P9V.harveyi *+++++++
SA26V.harveyi−+−+−++
P2V.alginolyticus *+++++++
P12V.hyugaensis++−−−+−
SA1A.veronii−−−+−++
SA15A.veronii *+++++++
SA17A.veronii *++++++−
SA25A.veronii *+−+++++
SA31A.veronii *+−++++−
SA3P.piscicida *−+−−++−
SA5P.damselae *−+−−++−
P5M.morganii+−−−−+−
P13S.indica *+++++++
SA9B.cereus *++++++−
SA7S.epidermidis−+++−++
SA21V.fluvialis *+++++++
(+): positive test, (−): negative test; * β-hemolytic strain.
Table
Table 3. Distribution of ARI in the different bacterial populations identified in this study.
Table 3. Distribution of ARI in the different bacterial populations identified in this study.
Microorganisms TestedAntibiotic Resistance Index (ARI)
Vibrio spp. (n = 9)0.542
Aeromonas spp. (n = 5)0.553
All Gram-negative0.544
All Gram-positive0.462
Table
Table 4. Exopolysaccharides production (slime) on Congo red agar, and ability to adhere to a glass surface (Wolfe test).
Table 4. Exopolysaccharides production (slime) on Congo red agar, and ability to adhere to a glass surface (Wolfe test).
CodeBacteria TestedSlime Production on CRAWolfe Test
MorphotypeInterpretation
P1V. natriegensRedNon producer++
P14V. natriegensBlackProducer+
SA11V. natriegensBlackProducer++
SA13V. natriegensOrangeNon producer++
SA27V. natriegensBlackProducer++
P9V. harveyiBlackProducer++
SA26V. harveyiRedNon producer+
P2V. alginolyticusBordeauxNon producer++
P12V. hyugaensisBordeauxNon producer+
SA1A. veroniiBlackProducer+
SA15A. veroniiBordeauxNon producer++
SA17A. veroniiWhite with red centerNon producer+
SA25A. veroniiBlackProducer++
SA31A. veroniiBlack grayNon producer+
SA3P. piscicidaRedNon producer+
SA5P. damselaeRedNon producer−
P5M. morganiiRedNon producer+
P13S. indicaBordeauxNon producer++
SA9B. cereusBordeauxNon producer++
SA7S. epidermidisRedNon producer+
SA21V. fluvialisBordeauxNon producer+
(++): large pellicule formation; (+) weak pellicule formation; (−) no pellicule formation.
Table
Table 5. Biofilm formation by the 21 identified bacterial strains from S. aurata and P. indicus on polystyrene 96-well plates, as well as glass and plastic abiotic surfaces.
Table 5. Biofilm formation by the 21 identified bacterial strains from S. aurata and P. indicus on polystyrene 96-well plates, as well as glass and plastic abiotic surfaces.
CodeBacteria TestedPolystyrene *Glass **Plastic **
OD595nm ± SDInterpretationOD595nm ± SDInterpretationOD595nm ± SDInterpretation
P1V. natriegens0.360 ± 0.024(−); Non biofilm forming0.102 ± 0.002(+); Weakly adherent0.152 ± 0.036(++); Moderately adherent
P14V. natriegens0.746 ± 0.002(−); Non biofilm forming0.114 ± 0.006(+); Weakly adherent0.412 ± 0.022(+++), Strongly adherent
SA11V. natriegens2.029 ± 0.166(++); Medium biofilm forming0.070 ± 0.004(−); Non adherent0.081 ± 0.013(+); Weakly adherent
SA13V. natriegens0.976 ± 0.061(−); Non biofilm forming0.108 ± 0.009(+); Weakly adherent0.387 ± 0.514(+++), Strongly adherent
SA27V. natriegens0.599 ± 0.026(−); Non biofilm forming0.189 ± 0.010(++); Moderately adherent0.148 ± 0.008(++); Moderately adherent
P9V. harveyi1.492 ± 0.119(+); Weak biofilm forming0.101 ± 0.027(+); Weakly adherent0.081 ± 0.017(+); Weakly adherent
SA26V. harveyi0.981 ± 0.178(−); Non biofilm forming0.245 ± 0.023(++); Moderately adherent0.219 ± 0.061(++); Moderately adherent
P2V. alginolyticus2.070 ± 0.076(++); Medium biofilm forming0.188 ± 0.045(++); Moderately adherent0.116 ± 0.033(+); Weakly adherent
P12V. hyuganesis2.029 ± 0.206(++); Medium biofilm forming0.075 ± 0.008(−); Non adherent0.067 ± 0.009(−); Non adherent
SA1A. veronii1.505 ± 0.072(+); Weak biofilm forming0.112 ± 0.007(+); Weakly adherent0.099 ± 0.022(+); Weakly adherent
SA15A. veronii1.500 ± 0.118(+); Weak biofilm forming0.140 ± 0.013(+); Weakly adherent0.055 ± 0.005(−); Non adherent
SA17A. veronii0.835 ± 0.055(−); Non biofilm forming0.091 ± 0.016(+); Weakly adherent0.077 ± 0.005(+); Weakly adherent
SA25A. veronii0.351 ± 0.021(−); Non biofilm forming0.158 ± 0.021(+); Weakly adherent0.060 ± 0.004(−); Non adherent
SA31A. veronii1.214 ± 0.216(+); Weak biofilm forming0.142 ± 0.010(+); Weakly adherent0.099 ± 0.005(+); Weakly adherent
SA3P. piscicida2.792 ± 0.244(++); Medium biofilm forming0.246 ± 0.027(++); Moderately adherent0.077 ± 0.001(+); Weakly adherent
SA5P. damselae0.410 ± 0.028(−); Non biofilm forming0.109 ± 0.010(+); Weakly adherent0.118 ± 0.019(+); Weakly adherent
P13S. indica0.505 ± 0.078(−); Non biofilm forming0.110 ± 0.007(+); Weakly adherent0.418 ± 0.004(+++), Strongly adherent
P5M. morganii0.782 ± 0.053(−); Non biofilm forming0.114 ± 0.012(+); Weakly adherent0.133 ± 0.009(+); Weakly adherent
SA9B. cereus2.525 ± 0.210(++); Medium biofilm forming0.313 ± 0.061(++); Moderately adherent0.151 ± 0.025(++); Moderately adherent
SA7S. epidermidis0.781 ± 0.023(−); Non biofilm forming0.114 ± 0.009(+); Weakly adherent0.182 ± 0.010(++); Moderately adherent
SA21V. fluvialis0.307 ± 0.011(−); Non biofilm forming0.116 ± 0.012(+); Weakly adherent0.078 ± 0.018(+); Weakly adherent
* Interpretation of biofilm formed on polystyrene surface [33]: (−) non biofilm forming OD595 ≤ 1; (+) weak biofilm forming 1 < OD595 ≤ 2; (++) medium biofilm forming 2 < OD595 ≤ 3; (+++) strong biofilm forming OD595 > 3. ** Interpretation of biofilm formed on glass and plastic surfaces [37]: nonadherent (0) OD ≤ ODc; weakly adherent (+) ODc < OD ≤ 2 × ODc; moderately adherent (++) 2 × ODc < OD ≤ 4 × ODc; strongly adherent (+++) 4 × ODc < OD.
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Abdulhakeem, M.A.; Alreshidi, M.; Bardakci, F.; Hamadou, W.S.; De Feo, V.; Noumi, E.; Snoussi, M. Molecular Identification of Bacteria Isolated from Marketed Sparus aurata and Penaeus indicus Sea Products: Antibiotic Resistance Profiling and Evaluation of Biofilm Formation. Life 2023, 13, 548. https://doi.org/10.3390/life13020548

AMA Style

Abdulhakeem MA, Alreshidi M, Bardakci F, Hamadou WS, De Feo V, Noumi E, Snoussi M. Molecular Identification of Bacteria Isolated from Marketed Sparus aurata and Penaeus indicus Sea Products: Antibiotic Resistance Profiling and Evaluation of Biofilm Formation. Life. 2023; 13(2):548. https://doi.org/10.3390/life13020548

Chicago/Turabian Style

Abdulhakeem, Mohammad A., Mousa Alreshidi, Fevzi Bardakci, Walid Sabri Hamadou, Vincenzo De Feo, Emira Noumi, and Mejdi Snoussi. 2023. "Molecular Identification of Bacteria Isolated from Marketed Sparus aurata and Penaeus indicus Sea Products: Antibiotic Resistance Profiling and Evaluation of Biofilm Formation" Life 13, no. 2: 548. https://doi.org/10.3390/life13020548

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