Abstract
To investigate the presence of Pseudomonas anguilliseptica, one hundred specimens of sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) were collected alongside water samples during episodes of widespread fish mortality in marine fish farms located in the northern region of Egypt. This study documented the clinical and postmortem manifestations observed in moribund sea bream and sea bass, thus providing evidence for the occurrence of a septicemic-hemorrhagic bacterial disease. Fourteen strains of P. anguilliseptica were isolated and characterized from both sea bream and sea bass specimens. The conventional bacteriology methods were employed to retrieve the causative bacterial agent and subsequently evaluate its phenotypic traits. Moreover, sequencing of the 16 S rRNA was conducted to characterize the identified microorganism. Furthermore, multilocus sequence analysis (MLSA) was employed to confirm the identity of P. anguilliseptica and elucidate the phylogenetic interrelationship among various strains. Regardless of their source or the fish species from which the strains were obtained, these isolates showed a high level of phenotypic homogeneity. MLSA displayed a genetic homogeneity among isolates despite their different geographic origins. Antibiogram revealed the sensitivity of some P. anguilliseptica strains for antibiotics (florfenicol, trimethoprim, sulfamethoxazole, cefotaxime, and ciprofloxacin). Isolated strains were harboring some antibiotic-resistant genes, with the most prevalent being tetA gene, followed by ermB gene. Water physico-chemical parameters (low temperature and salinity fluctuation) were convenient for bacterial growth. In addition, P. anguilliseptica strains could resist several antibiotics and harbored antibiotic-resistant genes, resulting in difficulties in fish treatment.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Pseudomonas species, a group of gram-negative bacteria, are widely distributed in aquatic environments and are known to be associated with stress-related diseases in farmed fish, particularly in intensive culture systems. These bacteria have been identified as the primary cause of fish diseases, leading to significant economic losses, with reported mortality rates reaching 100% in valuable fish species such as rainbow trout (Oncorhynchus mykiss), sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), and ayu (Plecoglossus altivelis) (López et al. 2012; Derome et al. 2016). The most commonly isolated species include Pseudomonas luteola, P. anguilliseptica, P. baetica, P. fluorescens, P. aeruginosa, P. koreensis, P. plecoglossicida, P. pseudoalcaligenes, P. putida, and P. chlororaphis (Austin and Austin 2016; Sherif et al. 2021).
P. anguilliseptica, a bacterium commonly found in aquatic environments, is characterized as a Gram-negative microorganism. It exhibits fastidious characteristics, displaying slow growth and weak reactions when cultured on bacterial media commonly employed in microbiology laboratories (Daly 1999; López-Romalde et al. 2003). P. anguilliseptica is an opportunistic microorganism responsible for causing a disease commonly referred to as red spot disease or “sekiten-byo” in Japan. This pathogen was initially documented in pond-farmed Japanese eel (Anguilla japonica) (Wakabayashi and Egusa 1972). Additionally, it is considered a prevalent infectious pathogen among farmed marine and brackish water fishes (Daly 1999). Outbreaks of P. anguilliseptica in farmed gilt-head seabream have been reported in several Mediterranean countries, including France, Portugal, and Spain. These outbreaks have resulted in a disease known as hemorrhagic septicemia, associated with high mortality rates and significant financial losses for the aquaculture industry (Doménech et al. 1999; Romalde et al. 2001; Ali et al. 2022). Similar occurrences have also been reported in other fish species such as turbot (Scophthalmus maximus), black sea bream (Acanthopagrus schlegeli), European eel (Anguilla anguilla), ayu (P. altivelis), sea trout (Salmo trutta), rainbow trout (O. mykiss), Atlantic salmon (Salmo salar), striped jack (Pseudocaranx dentex), whitefish (Coregonus sp.), Baltic herring (Clupea harengus membras), and orange-spotted grouper (Epinephelus coioides) (Al-Marzouk 1999; Austin and Austin 1999; Daly 1999; López-Romalde et al. 2002).
Identifying these bacterial species using microbiological analyses is challenging due to slow growth, low metabolic activity, and variability observed in their morphology and biochemical characteristics among different isolates. Consequently, molecular techniques accurately identify and distinguish P. anguilliseptica from other closely related genera (Mulet et al. 2012; Palleroni 2015). These techniques include 16 S rRNA sequencing for genus identification and multilocus sequence analysis (MLSA) for species classification (Mulet et al. 2012; Gomila et al. 2015; Abdelsalam et al. 2022). The multilocus sequence analysis (MLSA) technique was developed to assess molecular variations in multiple protein-coding housekeeping genes to compare closely related bacterial species (Mjølnerød et al. 2021). Multiple species of bacteria were subjected to standardization using MLSA, and the genetic variations derived from conserved housekeeping genes were revealed in the phylogenetic relationship. The MLSA technique is the most reliable method for studying the epidemiology, geographical distribution, and characterization of Pseudomonas spp. It can distinguish between different pathogenic strains of this pathogen (Sánchez et al. 2014; Gomila et al. 2015). The MLSA technique, which involves partial sequencing of four housekeeping genes (rpoB, rpoD, gyrB, and aroE), effectively identifies Pseudomonas spp. This technique has provided valuable insights into the phylogenetic relationships among different Pseudomonas strains and enabled discrimination between them (Mulet et al. 2012; Gomila et al. 2015).
Recently, antibiotic-resistant bacteria were frequently isolated from aquatic animals (Naik et al. 2018), aquatic water, and sediment (Pham et al. 2018; Shah et al. 2012). In Egypt, aquatic farmers and paramedics routinely use different antibiotics without a veterinary consultant during disease outbreaks, resulting in emerging antibiotic-resistant bacteria (WHO 2006; Sherif et al. 2021, 2022).
Over the past year, multiple reports of fish mortalities have occurred in farms that specifically reared seabass and seabream.
The objective of the current study was to conduct phenotypic and genetic characterizations of P. anguilliseptica strains isolated from moribund seabream and sea bass. Furthermore, the study aimed to investigate the weak response to antibiotic treatment. In addition, the quality of fish farm water associated with this particular bacterium was examined.
Materials and methods
Fish farms and samples collection
Specimens of moribund sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) were obtained from a collective of four fish farms located in the governorates of Dameitta (comprising three farms) and Kafrelsheikh (comprising one farm). These particular fish farms encountered high mortality rates during the winter season in November 2022. One hundred moribund fish were concurrently collected from affected fish ponds with water samples in triplicates, as described by Sherif et al. (2023) and Eldessouki et al. (2023). Water samples were collected at a depth of 0.5 m using aseptic techniques to guarantee the absence of contamination in the collected samples. The investigation was conducted to determine the presence of bacteria in fish samples. Bacterial cultures were obtained from the internal organs, specifically the kidney, spleen, and hepatopancreas tissues, using the protocols described by Woo and Bruno (2014) and Sherif and Kassab (2023).
Bacterial analyses
Bacterial isolation
Pooled specimens of livers, kidneys, and spleens, each fish species per farm considered one sample, were streaked on various agar mediums. Subsequently, these samples were incubated at 25 °C for 24 to 72 h (Wakabayashi and Egusa 1972). The agarmedia used consisted of tryptic soy agar (TSA) supplemented with 3% sodium chloride and 5% sheep blood agar. The prevailed colonies were selected and purified by subculturing onto TSA under the same conditions. Isolates were confirmed by selective isolation on Salmonella-Shigella agar SS agar. Gram staining, oxidase test, catalase activity, lactose fermentation on MacConkey agar, and motility testing using motility mediums were performed for the preliminary identification of bacterial isolates. The stock cultures were maintained at −70 °C in tryptic soy broth (Difco Laboratories), supplemented with 1% sodium chloride and 15% glycerol. The biochemical analyses were conducted in triplicate using API20 E following the guidelines provided by the manufacturer (BioMerieux, Marcy l’ Etoile, France) and the recommendations presented by Madigan and Martinko (2005). In this work, the biosafety protocols adhered to the Pathogen Safety Data Sheet for Infectious Substances-P. Anguilliseptica, as the Pathogen Regulation Directorate of the Public Health Agency of Canada (2022) outlines.
Molecular identification
DNA extraction
Genomic DNA from the pure 14 isolates of Pseudomonas spp. was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany, Cat. no. 513) following QIAGEN’s recommended protocol. The extracted DNA was eluted using the kit’s 100 µl elution buffer. The concentration and integrity of DNA were determined using the Nanodrop spectrophotometer following the manufacturer guidelines at 260/280 nm (ND-1000 UV-VIS, Thermo Scientific, USA). The extracted DNA was stored at − 20 °C for subsequent PCR amplifications and sequencing.
PCR amplifications
The identity of Pseudomonas spp. isolates was initially confirmed by amplifying and sequencing the 16 S rRNA gene using the universal primer pair 9 F and 1541R (Table 1), following the established protocol described by Osborne et al. (2005). For more in-depth phylogenetic analysis, four housekeeping genes—gyrB (703 bp), rpoB (529 bp), rpoD (766 bp), and aroE (653 bp)—were individually PCR amplified from each Pseudomonas isolate using multilocus sequence typing (MLST). The MLST primers were adopted from Mjølnerød et al. (2021). Additionally, bacterial strains were screened for resistance to tetracycline, erythromycin, sulfonamide, and quinolone by detecting the presence of resistance genes tetA, qnrs, sul1, and ermB, respectively, using PCR as described by Randall et al. (2004). All primers used in this study are documented in Table 1.
In brief, PCR amplifications were conducted in 25 µl total reaction volumes containing 4 µl DNA template (50 ng), 12.5 µl MyTaq Red Mix (Bioline), 1 µl of each primer (20 pmol), and 6.5 µl nuclease-free water. Each gene (16 S rRNA, gyrB, rpoB, rpoD, aroE, sul1, tetA, qnrs, and ermB) was individually amplified from every Pseudomonas isolate. Thermocycling conditions began with initial denaturation at 95 °C for 4 min followed by 35 cycles of denaturation at 95 °C for 1 min, annealing for 1 min, and extension at 72 °C for 1 min. Annealing temperatures were adjusted based on the target gene, specifically 52 °C for 16 S rRNA, 56 °C for the four MLST genes gyrB, rpoB, rpoD, and aroE, 55 °C for tetA, 68 °C for sul1, 53 °C for qnrs, and 62 °C for ermB. A final extension step at 72 °C for 7 min completed the PCR. Amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) per the manufacturer’s instructions. Purified products were Sanger sequenced in both forward and reverse directions using the ABI 3730xl DNA Analyzer (Applied Biosystems). Raw sequence reads were trimmed, edited, and assembled into contigs using BioEdit version 7.2.5 (Hall 1999).
For species identification, the assembled 16 S rRNA sequences were aligned against other 16 S rRNA sequences of Pseudomonas spp. using the online BLAST tool of NCBI. The obtained 14 sequences were deposited in the database of GenBank under the following succession accession numbers starting from ON926995 to ON927008 (Tables 2 and 3). The identity of Pseudomonas isolates was confirmed at the species level when the BLAST similarity reached ≥ 99 % to the relevant 16 S rRNA sequences available in the GenBank database, as illustrated by Drancourt et al. (2000). The multiple sequence alignments were carried out using the MEGA program to match the interspecies and intrastrain similarities index (Kumar et al. 2018). The neighbor-joining phylogenetic analysis of the current 16 S rRNA sequences was performed against 14 typing strains including, P. anguilliseptica (3 isolates), P. resinovorans (1 isolate), P. aeruginosa (1 isolate), P. asplenii (1 isolate), P. monteilii (1 isolate), P. tolaasii (1 isolate), P. fluorescens (2 isolates), P. chlororaphis (2 isolates), and P. putida (2 isolates).
Multilocus sequence analysis
The raw sequences of gyrB, rpoB, rpoD, and aroE genes were trimmed and edited using BioEdit software ver. 7.2.5 (Hall 1999). The assembled sequences were submitted to the GenBank database under the following accession numbers: gyrB (OQ694532- OQ694545), rpoB (OQ726020- OQ726033), rpoD (ON981406- ON981419), and aroE (OQ744508- OQ744521). The final sequences were imported and concatenated in Microsoft Excel. The phylogenetic analysis of the concatenated sequences of housekeeping genes was constructed by neighbor-joining methods of the MEGA 11 with the pairwise deletion option (Kumar et al. 2018). The node supports were estimated using nonparametric bootstrapping with 1000 replicates and Kimura’s two-parameter model was applied.
Antibiogram of Pseudomonas anguilliseptica strains
In line with an earlier study (CLSI 2010) and using the disc diffusion method, bacterial strains were examined for their sensitivity to some antibiotics on Mueller-Hinton agar (Oxoid™). The antimicrobial agents utilized in the assessment were tetracycline (TetA) 30 µg, ciprofloxacin (Cip) 5 µg, trimethoprim/sulfamethoxazole (SXT) 1.25/23.75 µg, erythromycin (E, 15 µg), florfenicol 30 µg, gentamicin 10 µg, amoxicillin 10 µg, kanamycin 30 µg, cefotaxime 30 µg, ampicillin (Amp) 10 µg, and streptomycin 30 µg. The test was conducted in triplicates, and isolates of P. anguilliseptica were subcultured into tryptic soy broth, then incubated overnight at 30 °C, and placed onto Mueller-Hinton agar plates using a cotton swab. Following incubation for 24 h at 30 °C, the diameter of the inhibition zones was measured, and the results were interpreted based on the Clinical Laboratory Standards Institute criteria. The equation determined the multidrug resistance (MDR) index:
x represents the number of antibiotic categories to which the isolates were resistant, while y represents the assessment of antibiotics. If the obtained MDR index is greater than 0.2, the bacterial strain is resistant to multiple antibiotics (Krumperman 1983).
Examination of water physicochemical parameters
The temperature and salinity of water samples at farm sites were analyzed using a YSI Environmental (Model EC300), while dissolved oxygen (DO) was analyzed using an Aqualytic (Model OX24), and pH was analyzed using a Thermo Orion (Model 420 A). The samples were collected in 1-L polyethylene bottles and transported on ice to the laboratory, namely, total ammonia nitrogen (TAN), unionized ammonia (NH3), nitrite (NO2), and nitrate (NO3); a UV/visible spectrophotometer (Thermo-Spectronic 300) was used for ammonia compound analyses following the recommendations of Rice and Bridgewater (2012).
Statistical examination
Different parameters were assessed using the ANOVA test and Duncan’s Multiple Range (Duncan 1955) by determining the mean and standard error of the collected data using SPSS software (SPSS 2004). At P ≤ 0.05, values were considered statistically significant.
Results
Clinical signs and post-mortem of farmed sea bream and sea bass infected with P. anguilliseptica
In Fig. 1, the moribund sea bream showed signs of septicemic bacterial infection. These were hemorrhages on the external body surface behind the pectoral fin and abdomen with ulcers under the dorsal fin. Postmortem signs were hemorrhages on the liver surface and splenomegaly (rounded surface).
In Figs. 2 and 3, moribund sea bass showed hemorrhages on the fish skull, eye loss, hemorrhages on the external body surface, and gills with faint red. Post-mortem revealed signs of hemorrhagic diseases: light-brownish liver, hemorrhages on liver and internal surfaces, abdominal fat with splenomegaly, and hemorrhagic-distended kidney.
Bacterial growth on agar plate
The isolated bacteria were negative for gram stain, and by using API 20E, they had the number 220,004, which represented the Pseudomonas spp. The bacterial growth was evaluated in different incubation temperatures (8, 25, 37, and 42 °C) and salinity (0, 1, 3, 5 g/L) (Table 2). No growth was detected in all bacterial strains at 37 and 42 °C, while NDA0208 and NDA0209 (both are from Damietta farms) and all bacterial isolates of Kafrelshiekh farms (NKF0301-5) showed weak growth. All P. anguilliseptica strains were unable to grow in 0 g/L salinity. Also, all strains isolated from Damietta farms showed weak growth in 1 g/L salinity, while those recovered from Kafrelsheikh did not grow. All strains could grow in 3 and 5 g/L, except those recovered from Kafrelsheikh had weak growth in 5 g/L.
Sequencing of 16 S rRNA and MLST
The amplified 16 S rRNA gene was successfully performed from 14 Pseudomonas spp. isolates using the gene-specific primers pair. The purified PCR products were then sequenced to confirm the identities of Pseudomonas spp. at the species level. The successive accession numbers of these sequences (ON926995 to ON927008) were issued by the GenBank staff (Table 3). The BLAST analysis of 16 S rRNA gene sequences proved that all 14 bacterial strains belonged to the genus Pseudomonas, and all isolates were identified as P. anguilliseptica. The intrastrain nucleotide differences within the isolates of P. anguilliseptica varied from 99.80 to 99.39% with three to nine nucleotide differences. The comparative sequence analysis of the current 16 S rRNA sequences (ON926995 to ON927008) revealed a high similarity index ranging between 99.86 and 99.39% with relevant sequences of P. anguilliseptica (AB021376T, LC071931, LC194236, LC071928, LC071927, and LC071926). The alignment of these accession numbers (ON926995 to ON927008) showed 96.73–95.45% 16 S rRNA similarity to typing isolates of the following Pseudomonas spp.: P. resinovorans (NR_112062) T, P. aeruginosa (AY268175) T, P. asplenii (NR_040802) T, P. monteilii (KU057954) T, P. tolaasii (NR_114481) T, P. fluorescens (AF094731) T, P. chlororaphis (FJ652608) T, and P. putida (AF094743) T.
In Fig. 4, the phylogenetic analysis of the 16 S rRNA sequences of P. anguilliseptica isolates formed two major clades. The first clade was separated into two subclades. The first subclade involved all P. anguilliseptica, grouped with the sequence of typing P. anguilliseptica (NCIMB 1949) with a substantial 100% bootstrap value. In this study, all isolates of P. anguilliseptica were separated from other Pseudomonas spp. belonging to P. resinovorans, P. aeruginosa, P. asplenii, P. monteilii, P. tolaasii, P. fluorescens, P. chlororaphis, and P. putida. The typing strain of S. dysgalacatiae (ATCC 43,078) served as an outgroup.
In Table 3, MLSA allowed unambiguous identification of all recovered Pseudomonas spp. isolates as P. anguilliseptica (14 isolates). All isolates shared 99.62–99.70% concatenated sequence similarity to typing P. anguilliseptica (NCIMB 1949) strain. In addition, the alignment of concatenated sequences of these isolates showed high discriminatory power between P. anguilliseptica of this study and typical isolates of P. chlororaphis subsp. piscium ATCC 17,809 (CP027709.1)T, P. fluorescens ATCC 13,525 (QVNA00000000.1)T, P. aeruginosa ATCC 27,853 (CP015117.1)T, and P. putida ATCC 12,633 (CP101910.1)T, with similarity index ranging between 80.92 and 79.54%. On the other hand, the comparative sequence alignment of four concatenated housekeeping genes (2363 bp) demonstrated similarity scores ranging between 99.75 and 100% among all retrieved isolates.
In Fig. 5, the phylogenetic tree derived from MLSA based on concatenated sequences of 14 isolates displayed five distinct sequence types (ST1, ST2, ST3, ST4, and ST5) (Fig. 2). The phylogeny tree of MLSA revealed that all P. anguilliseptica isolates belonged to three main clusters. Clustering was correlated with the geographical source of the strains. Clusters 1 and 2 were associated with isolates originating from Damietta farms, while cluster 3 was related to isolates recovered from Kafrelsheikh farms. The first clade grouped ST1 and ST2, and the node supported with a bootstrap value of 73%. The second clade gathered ST3, with a bootstrap value of 98%. The third clade grouped ST4 and ST5 and supported with a bootstrap value of 96%.
Antibiotic resistance genes and antibiogram of P. anguilliseptica strains
Concerning the prevalence of ARG, tetracycline (tetA) was the most predominant, followed by erythromycin (ermB), while sulfonamide (sul1) and quinolone (qnrs) did not present in those isolated at Kafrelsheikh farm (Tables 4 and 5).
In Table 4, P. anguilliseptica strains were tested against 11 antibiotic types. All P. anguilliseptica strains were resistant to the selected antibiotics tetracycline, amoxicillin, ampicillin, and kanamycin, whereas they were sensitive to gentamycin, florfenicol, and trimethoprim plus sulfamethoxazole. Antibiogram of P. anguilliseptica strains differed according to the site of collection. A set of tetracycline (TE), ampicillin (AMP), amoxicillin (AML), kanamycin (K), and E was predominant in P. anguilliseptica trimethoprim/sulfamethoxazole (SXT), ciprofloxacin (Cip), and streptomycin (S) not in NKF were presented in those recovered at Damietta farms and absent at Kafrelsheikh farm (Table 5).
Examination of water physicochemical parameters of fish farms
In Table 6, water temperature ranged between 18.03 and 19.87 °C, showing significant differences suitable for bacterial infection. The water salinity was significantly higher in Damietta farm (22.8 to 225 g/L) than Kafrelshiekh farm (19.43 g/L). The level of DO was above 4 mg/L in all farms except farm ND2 at Damietta, which was 3.43 mg/L. The pH levels, which ranged between 8.17 and 8.22, were suitable for fish culture and bacterial growth. Ammonia compounds, both TAN and total ammonium (NH3 + NH4), were significantly higher in ND2 and ND3 compared to investigated farms (0.56 and 0.58; 0.72 and 0.74 mg/L, respectively). With the same trend, NH3 was significantly higher in ND2 and ND3 (0.03 and 0.037 mg/L, respectively). There were no significant differences in NO2 and NO3 in fish farms under investigation.
Discussion
Clinical and post-mortem signs were recorded in moribund sea bream and sea bass in this study, indicating the occurrence of septicemic-hemorrhagic bacterial disease, which were hemorrhages on the external body surface and internal organs with faint-reddish gills, splenomegaly, and hemorrhagic-distended kidney. In the field studies, general clinical symptoms such as pale skin color, loss of scales, hemorrhages, and ulcers on the body surface, and anemia in the internal organs were observed and showed similarities with previous reports for each particular bacterial fish pathogen (Akayli 2001; Austin and Austin 2016; Roberts 2012).
In this work, microbiological methods of isolation and identification confirm the presence of gram–ve, and identification number 220,004 was obtained using API 20E, indicating the presence Pseudomonas spp. unable to identify the bacteria associated with sea bream and sea bass mass mortality. Similarly, false-negative results were detected in the API 20NE (Balboa et al. 2007), with P. anguilliseptica (López-Romalde 2005; Canak and Akayli (2018). Even when Topic Popovic et al. (2007) developed API 20E kits for pathogenic fish bacteria used in field, they recorded misidentification with P. stutzeri and P. anguilliseptica isolated from moribund gilt-head sea bream. In addition, P. anguilliseptica was misidentified as P. fluorescens/putida with API 20NE kit Canak and Akayli (2018). So, the API 20NE method could be used to screen and confirm the presence of Pseudomonas spp. in sea bream and sea bass.
In this work, no bacterial growth was detected in all bacterial strains at 37 and 42 °C, and no growth was detected at 0 g/L salinity. All strains could grow in 3 and 5 g/L, except those recovered from Kafrelsheikh had weak growth in 5 g/L. Similarly, Pseudomonas spp. could tolerate a wide range of water temperature, pH, and salinity and thus could remain in the aquatic environment; however, nutrient sources are scarce (Liu et al. 2015; Palleroni 2015; Moradali et al. 2017). Inconsistency, Duman et al. (2021) found that Pseudomonas spp. could grow at NaCl concentrations of 0–45 g/L, whereas higher salinity 60, 70, 80, and 90 g/L NaCl resulted in different growth rates of 20%, 61%, 73%, and 91%, respectively, such as strains of P. haemolytica, P. proteolytica, P. lundensis, and P. weihenstephanensis. In addition, it was noted that all Pseudomonas spp. could grow at 37 °C incubation temperature, such as P. mandelii, P. meridiana, P. migulae, and Pseudomonas spp. tolerated 42 °C, and P. haemolytica was heat-tolerant up to 45 °C. It can be easily differentiated Pseudomonas sp. from P. anguilliseptica based on its ability to grow at 41 °C, which P. anguilliseptica cannot (Austin and Austin 1999).
Due to the misidentification of Pseudomonas sp. using traditional bacteria methods, ninety isolates were identified using the phylogenetic method with 4-gene multilocus sequence analysis based on the partial sequencing of the 16 S rRNA, gyrB, rpoB, and rpoD genes (Duman et al. 2021).
In this study, sequencing of 16 S rRNA confirmed that all 14 bacterial strains belonged to the genus Pseudomonas group. By BLAST analysis of 16 S rRNA, Pseudomonas strains were molecularly recognized as P. anguilliseptica (14 isolates).
However, relying on the 16 S rRNA could lead to misidentification in some instances (Elgendy et al. 2022). In this study, sequencing of 16 S rRNA allowed the differentiation of P. anguilliseptica isolates from other typing Pseudomonas spp. isolates of P. resinovorans, P. aeruginosa, P. asplenii, P. monteilii, P. tolaasii, P. fluorescens, P. chlororaphis, and P. putida and showed 96.73–95.45% similarity score index. Mulet et al. (2012) and Gomila et al. (2015) obtained similar results.
On the other hand, the MLSA assay based on four housekeeping genes (gyrB, rpoB, rpoD, and aroE) was done to confirm the identification of isolates and to study the phylogenetic relationships in P. anguilliseptica. In addition, several studies used these five housekeeping genes (16 S rRNA, gyrB, rpoB, rpoD, and aroE) to characterize and epidemiological studies of Pseudomonas spp. In this study, the concatenation of four housekeeping genes provided sufficient discriminatory power to separate P. anguilliseptica isolates regarding sequence divergences from other Pseudomonas spp. A prominent gap between the intraspecies and interspecies MLSA similarities is essential for identifying the isolates at the species level, and a threshold of 97% of MLSA similarity was suggested for species differentiation in the genus Pseudomonas (Mulet et al. 2010, 2012). In this study, P. anguilliseptica isolates shared the highest MLSA similarity (99.62–99.70%) with the typing P. anguilliseptica (NCIMB 1949) and displayed 80.92–79.54% MLSA similarity index with other typing isolates of Pseudomonas spp.: P. chlororaphis subsp. piscium ATCC 17,809 (CP027709.1)T, P. fluorescens ATCC 13,525 (QVNA00000000.1)T, P. aeruginosa ATCC 27,853 (CP015117.1)T, and P. putida ATCC 12,633 (CP101910.1)T. Therefore, the MLSA assay of four HKGs (gyrB, rpoB, rpoD, and aroE) revealed a considerable performance for the identification of P. anguilliseptica. On the other hand, MLSA is superior for resolving the intrastrain relationship among P. anguilliseptica isolates (Mjølnerød et al. 2021).
In this study, P. anguilliseptica strains were tested against 11 antibiotic types. All P. anguilliseptica strains were resistant to the selected antibiotics tetracycline, amoxicillin, ampicillin, and kanamycin, while they were sensitive to gentamycin, florfenicol, and trimethoprim plus sulfamethoxazole. Antibiogram of P. anguilliseptica strains differed according to the fish collection site. The most predominant ARG was tetA followed by ermB and sul1. There was a good correlation between the presence of resistance genes and corresponding resistance phenotypes, suggesting resistance genes, when present, were usually expressed (Randall et al. 2004). Accordingly, the antibiotics used in antibiogram tests were available in the local market; they could also be used in fish treatment (Noga 2010). Similarly, Canak and Akayli (2018) found that the isolated P. anguilliseptica was sensitive to ciprofloxacin and enrofloxacin, whereas it was resistant to 6 of the 10 antimicrobials. Other reports considered Pseudomonas spp. multidrug-resistant bacteria as they were resistant to ampicillin, trimethoprim + sulfamethoxazole, cefotaxime, aztreonam, and nitrofurantoin (Matyar et al. 2008; Devarajan et al. 2017); also, López-Romalde et al. (2003) found that all P. anguilliseptica strains were trimethoprimsulfomethoxazol. In contrast, Haenen and Davidse (2001) recorded that P. anguilliseptica were sensitive to neomycine, trimethoprim, trimethoprimqsulphadiazine, sulphonamids, oxytetracycline, flumequine, and oxolinic acid. These differences are attributed to use or lack of the use of these antimicrobials.
The intensive culture of sea bream in Egypt resulted in a high infection rate of pathogenic bacteria that grow well in brackish water ponds 23.5 to 26.5 g/L (Eisawy and Wassef 1984). In this work, water temperature ranged between 18.03 and 19.87 °C, while salinity was significantly higher in Damietta (22.8 to 22.5 g/L) than in Kafrelshiekh farm (19.43 g/L); these parameters were suitable for bacterial infection. Similarly, Haenen and Davidse (2001) isolated P. anguilliseptica at 23–25 °C in diseased European elvers that were maintained in Netherlands. These findings could be due to the decline in antibody production. Accordingly, Yamagachi et al. (1980) found that fish were immunosuppressed in response to a decline in water temperature and salinity because of a decrease in antibody formation as antibody production was seasonality dependent. In addition, the fluctuation of water temperature suppressed the fish immunity, causing them to become more vulnerable to P. anguilliseptica infection (Tort et al. 1996), so mortality ceased by raising the water temperature above 26 °C (Muroga et al. 1973; Haenen and Davidse 2001). Accordingly, bacterial disease outbreaks were recorded in fish farms at a water temperature of 16–18 °C (Kim et al. 2010), whereas the disease persisted in sea bream until the water temperature increased to 18–20 °C (Doménech et al. 1999).
The level of NH3 was stressful for fish in the examined fish farms according to standard values recommended (0.002–0.05 mg/L) by FAO (2023). Ammonia compounds were not statistically correlated with isolated bacteria strains, antibiotic resistance, MDR index, or ARGs. Accordingly, the ammonia compounds accumulated did not significantly affect the bacterial community or ARG composition; they might still cause immunosuppression status in the fish and other metabolic processes. These effects could render the fish more vulnerable to bacterial infections (Sreedevi and Ramasubramanian 2011; Rousselet et al. 2018; Sherif and AbuLeila 2022). Other findings, it was found that P. anguilliseptica infection is interrelated with winter disease (WD) syndrome in sea bream (Doménech et al. 1999); this syndrome is a multifactorial disease associated with many factors, mainly deteriorated water quality (high ammonia compound and heavy metals pollution) which could cause immunosuppressed fish (Tort et al. 1998).
Conclusion
In this study, MLSA was used to type fourteen P. anguilliseptica isolates. Bacterial infections were accompanied by low water temperature and fluctuation of water salinity. Despite the stress levels of unionized ammonia (NH3) in fish farm water, no statistical correlation was observed with bacterial infection. All P. anguilliseptica strains could grow in 3 and 5 g/L, except those recovered from Kafrelsheikh farm, which had weak growth in 5 g/L. Isolates were sensitive to florfenicol, trimethoprim, sulfamethoxazole, cefotaxime, and ciprofloxacin. In addition, tetracycline tetA and erythromycin ermB genes were the highest-presentedantibiotic-resistant genes.
Data availability
The datasets and materials used in this study are available in the manuscript.
References
Abdelsalam M, Elgendy MY, Elfadadny MR, Ali SS, Sherif AH, Abolghait SK (2022) A review of molecular diagnoses of bacterial fish diseases. Aquac Int 31:417–434. https://doi.org/10.1007/s10499-022-00983-8
Akayli T (2001) Identification of vibriosis in cultured gilthead seabream (Sparus aurata, L 1758) by using ELISA and bacteriological methods. Istanbul University, Institute of Science, Ph.D. Thesis
Al-Marzouk AE (1999) Association of Pseudomonas anguilliseptica with mortalities in cultured marine orange-spotted grouper Epinephelus coioides in Kuwait. Fish Pathol 34:167–168
Ali NG, Ali TES, Kamel MF, Ibrahim RS, Sherif AH, Aboyadak IM (2022) Eradication of Livoneca redmanii infestation in cultured Argyrosomus regius. Aquaculture 558:738373. https://doi.org/10.1016/j.aquaculture.2022.738373
Austin B, Austin DA (1999) Bacterial fish pathogens: disease of farmed and wild fish, 3rd edn. Springer, Heidelberg
Austin B, Austin DA (2016) Pseudomonads. Bacterial fish pathogens. Springer International Publishing, pp 475–498. https://doi.org/10.1007/978-3-319-32674-0_9
Balboa S, Ferguson HW, Romalde JL (2007) Phenotypic, serological and genetic characterization of Pseudomonas anguilliseptica strains isolated from Cod, Gadus morhua L., in northern Europe. J Fish Dis 30(11):657–664
Canak O, Akayli T (2018) Bacteria recovered from cultured gilt-head seabream (Sparus aurata) and their antimicrobial susceptibilities. Eur J Biol 77(1):11–17
CLSI (2010) Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, Twentieth Informational Supplement M100-S20, CLSI, Wayne, Pa, USA. 2010
Daly JG (1999) Other bacterial pathogens. In: Woo PTK, Bruno DW (eds) Fish dis-eases and disorders: volume 3: viral, bacterial and fungal Infections. CABI Publishing, London, New York, pp 577–598
Derome N, Gauthier J, Boutin S, Llewellyn M (2016) The rasputin effect: when commensals and symbionts become parasitic. Advances in Environmental Microbiology Vol. 3. Springer, Berlin. https://doi.org/10.1007/978-3-319-28170-4
Devarajan N, K¨ohler T, Sivalingam P, van Delden C, Mulaji CK, Mpiana PT, Ibelings BW, Pot´e J (2017) Antibiotic resistant Pseudomonas spp. in the aquatic environment: a prevalence study under tropical and temperate climate conditions. Water Res 115:256–265. https://doi.org/10.1016/j.watres.2017.02.058
Doménech A, Frenández-Garayzabal JF, Garc´ıa JA, Cutuli MT, Blanco M, Gibello A, Moreno MA, Dom´ınguez L (1999) Association of Pseudomonas anguilliseptica infection with winter disease in sea bream (Sparus aurata). J Fish Dis 22:69–71
Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D (2000) 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J C Microbiol 38:3623–3630
Duman M, Mulet M, Altun S et al (2021) The diversity of Pseudomonas species isolated from fish farms in Turkey. Aquaculture 535:736369
Duncan (1955) Multiple range and multiple F test. Biometrics 11:10
Eisawy AM, Wassef E (1984) Preliminary studies on rearing of the gilthead sea bream, S. Aurata, in brackish water ponds. Aquaculture 38:255–260
Eldessouki EA, Salama SSA, Mohamed R, Sherif AH (2023) Using nutraceutical to alleviate transportation stress in the Nile tilapia. Egypt J Aquat Biol Fish 27(1):413–429. https://doi.org/10.21608/ejabf.2023.287741
Elgendy MY, Sherif AH, Kenawy A, Abdelsalam M (2022) Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities. Microb Pathog 169:105620
FAO (2023) Water, a rearing environment and its management in aquaculture. https://www.fao.org/3/af011e/AF011E06.HTM
Gomila M, Pe˜na A, Mulet M, Lalucat J, García-Vald´es E (2015) Phylogenomics and systematics in Pseudomonas. Front Microbiol 6:1–13. https://doi.org/10.3389/fmicb.2015.00214
Haenen OLM, Davidse A (2001) First isolation and pathogenicity studies with Pseudomonas anguilliseptica from diseased European eel Anguilla anguilla žL.in the Netherlands. Aquaculture 196:27–36
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98
Kim SR, Park M, Kitamura SI, Jung SJ, Oh MJ (2010) Recovery of Pseudomonas anguilliseptica from diseased striped beakperch (Oplegnathus fasciatus) in Korea. Fish Aqua Sci 13(2):190–194
Krumperman PH (1983) Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol 46(1):165–170. https://doi.org/10.1128/aem.46.1.165-170.1983
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol evol 35(6):1547
López JR, Di´eguez AL, Doce A, de la Roca E, de la Herran R, Navas JI, Toranzo AE, Romalde JL (2012) Pseudomonas baetica sp. nov., a fish pathogen isolated from wedge sole, Dicologlossa cuneata (Moreau). Int J Syst Evol Microbiol 62:874–882. https://doi.org/10.1099/ijs.0.030601-0
Liu Y, Rzeszutek E, Van Der Voort M, Wu CH, Thoen E, Skaar I, Bulone V, Dorrestein PC, Raaijmakers JM, De Bruijn I (2015) Diversity of aquatic Pseudomonas species and their activity against the fish pathogenic oomycete Saprolegnia. PLoS One 10:1–17. https://doi.org/10.1371/journal.pone.0136241
López-Romalde S (2005) Caracterizacio´n del Pato´geno Emergente en Acuicultura Marina Pseudomonas anguilliseptica. PhD thesis, Universidad de Santiago de Compostela, Spain
López-Romalde S, Magariños B, Ravelo C, Toranzo AE, Romalde JL (2002) Development of a PCR-based procedure for the rapid detection of Pseudomonas anguilliseptica. In: Proceedings of the Fourth International Symposium on Aquatic Animal Health, New Orleans, LO, USA, p 144
López-Romalde S, Nú˜nez S, Toranzo AE, Romalde JL (2003) Black spot seabream (pag-ellus bogaraveo), a new susceptible host for Pseudomonas anguilliseptica. BullEur Assoc Fish Pathol 23:258–264
Madigan MT, Martinko J (2005) Brock biology of microorganisms, 11th edn. Prentice Hall, Hoboken
Matyar F, Kaya A, Dinçer S (2008) Antibacterial agents and heavy metal resistance in gram-negative bacteria isolated from seawater, shrimp and sediment in Iskenderun Bay. Turk Sci Total Environ 407:279–285. https://doi.org/10.1016/j.scitotenv.2008.08.014
Mjølnerød EB, Nilsen HK, Gulla S, Riborg A, Bottolfsen KL, Wiklund T, Christiansen D, López Romalde JÁ, Scholz F, Colquhoun DJ (2021) Multilocus sequence analysis reveals different lineages of Pseudomonas anguilliseptica associated with Disease in farmed lumpfish (Cyclopterus lumpus L). PLoS One 16(11):e0259725
Moradali MF, Ghods S, Rehm BHA (2017) Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol 7. https://doi.org/10.3389/fcimb.2017.00039
Mulet M, Lalucat J, García-Valdés E (2010) DNA sequence-based analysis of the Pseudomonas species. Environ Microbiol 12:1513–1530
Mulet M, Gomila M, Scotta C, S´anchez D, Lalucat J, García-Vald´es E (2012) Concordance between whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the Genus Pseudomonas. Syst Appl Microbiol 35:455–464. https://doi.org/10.1016/j.syapm.2012.08.007
Muroga K, Jo Y, Yano M (1973) Studies on red spot disease of pond-cultured eels-I. The occurrence of the disease in eel culture ponds in Tokushima prefecture in 1972. Fish Pathol 8:1–9
Naik OA, Shashidhar R, Rath D, Bandekar JR, Rath A (2018) Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India. Environ Sci Pollut Res 25(7):6228–6239. https://doi.org/10.1007/s11356-017-0945-7
Noga EJ (2010) Pharmacopoeia. Fish disease: diagnosis and treatment, 2nd edn. Wiley, Hoboken, pp 375–420
Osborne CA, Galic M, Sangwan P, Janssen PH (2005) PCR-generated artefact from 16S rRNA gene-specific primers. FEMS Microbiol let 248(2):183–187
Palleroni NJ (2015) Pseudomonas, Bergey’s manual of systematics of archaea and bacteria. Wiley, North Caldwell. https://doi.org/10.1002/9781118960608.gbm01210
Pham TTH, Rossi P, Dinh HDK, Pham NTA, Tran PA, Ho TTKM, Dinh QT, De Alencastro LF (2018) Analysis of antibiotic multi-resistant bacteria and resistance genes in the effluent of an intensive shrimp farm (Long An, Vietnam). J Environ Manage 214:149–156. https://doi.org/10.1016/j.jenvman.2018.02.089
Public Health Agency of Canada (2022) The Honourable Leona Aglukkaq, P.C., M.P. Minister of Health. https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines/third-edition.html
Randall LP, Cooles SW, Osborn MK, Piddock LJV, Woodward MJ (2004) Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J Antimicrob Chemother 53(2):208–216. https://doi.org/10.1093/jac/dkh070
Rice EW, Bridgewater L (2012) A.P.H. Association, standard methods for the examination of water and wastewater, American Public Health Association, Washington, DC
Roberts R (2012) Fish pathology. 4th edn. Wiley-Blackwell, Hoboken
Robicsek A, Strahilevitz J, Sahm DF, Jacoby GA, Hooper DC (2006) Qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates fromthe United States. Antimicrob Agents Chemother 50(8):2872–2874. https://doi.org/10.1128/AAC.01647-05
Romalde JL, López-Romalde S, Magariños B, Núñez S, Toranzo AE (2001) Phenotypic characterization of Spanish isolates of Pseudomonas anguilliseptica causing winter disease in sea bream. In: Proceedings of the X International Conference on the European Association of Fish Pathologists (EAFP), Dublin, Ireland, p 22
Rousselet E, Stacy NI, Rotstein DS, Waltzek TB, Griffin MJ, Francis-Floyd R (2018) Systemic edwardsiella tarda Infection in a western African lungfish (protopterus annectens) with cytologic observation of heterophil projections. J Fish Dis 41(9):1453–1458
Sánchez D, Mulet M, Rodríguez AC, David Z, Lalucat J, García-Valdés E (2014) Pseudomonas aestusnigri sp. nov., isolated from crude oil-contaminated intertidal sand samples after the prestige oil spill. Syst Appl Microbiol 37:89–94
Shah SQ, Colquhoun DJ, Nikuli HL, Sorum H (2012) Prevalence of antibiotic resistance genes in the bacterial flora of integrated fish farming environments of Pakistan and Tanzania. Environ Sci Technol 46(16):8672–8679. https://doi.org/10.1021/es3018607
Sherif AH, AbuLeila RH (2022) Prevalence of some pathogenic bacteria in caged- Nile Tilapia (Oreochromis Niloticus) and their possible treatment. Jordan J Biol Sci 15(2):239–247. https://doi.org/10.54319/jjbs/150211
Sherif AH, Kassab AS (2023) Multidrug-resistant Aeromonas bacteria prevalence in Nile tilapia broodstock. BMC Microb 23(1):80. https://doi.org/10.1186/s12866-023-02827-8
Sherif AH, Gouda M, Darwish S, Abdelmohsin A (2021) Prevalence of antibiotic-resistant bacteria in freshwater fish farms. Aquac Res 52(5):2036–2047. https://doi.org/10.1111/are.15052
Sherif AH, Prince A, Adel Seida A, Saad Sharaf M, Eldessouki EA, Harfoush MA (2022) Moringa oleifera mitigates oxytetracycline stress in Oreochromis niloticus. Aquac Res 53(5):1790–1799. https://doi.org/10.1111/are.15707
Sherif AH, Eldessouki EA, Sabry NM, Ali NG (2023) The protective role of iodine and MS-222 against stress response and bacterial infections during Nile tilapia (Oreochromis niloticus) transportation. Aquac Int 1–16. https://doi.org/10.1007/s10499-022-00984-7
SPSS (2004) Statistical and package for social science, SPSS for windows release14.0.0, 19 June, 2004. Standard Version, Copyright SPSS Inc., pp 1989–2004
Sreedevi PR, Ramasubramanian V (2011) Biocontrol of ammonia pollution in the rearing water of fish by inducing a heterotrophic bacterial-based food chain in the medium. Aquac Int 19(4):789–796
Topic Popovic N, Coz-Rakovac R, Strunjak-Perovic I (2007) Commercial phenotypic tests (API 20E) in diagnosis of fish bacteria: a review. Vet Med 52:49–53
Tort L, Gomez E, Montero D, Sunyer JO (1996) Serum hemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies. Aqua Int 4:31–41
Tort L, Padrós F, Rotllant J, Crespo S (1998) Winter syndrome in the gilthead sea bream Sparus aurata. Immunological and histopathological features. Fish Shellfish Immunol 8:37–47
Wakabayashi H, Egusa S (1972) Characteristics of a Pseudomonas sp. from an epizootic of pond-cultured eels (Anguilla japonica) Bull Jpn Soc Sci Fish 38(6):577–587
WHO, World Health Organization (2006) Report of a joint FAO/OIE/WHO Expert Consultation on antimicrobial use in aquaculture and antimicrobial resistance. Seoul, Republic of Korea, 13–16 June 2006
Woo P, Bruno D (2014) Diseases and disorders of finfish in cage culture, 2nd edn. CABI Pub, Oxon, pp 159–160
Yamagachi N, Teshima C, Kurashige S, Saito T, Mitsuhashi S (1980) Seasonal modulation of antibody formation in fish (Salmo gairdneri) against heterologous erythrocytes and bacterial antigen. In: Solomon JB (ed), Aspects of developmental and comparative immunology vol 1. Fergamon Press, Oxford, pp 483-5
Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Author information
Authors and Affiliations
Contributions
This study was conducted in cooperation with all authors.
Corresponding authors
Ethics declarations
Ethics approval
This study was approved by the Ethics Committee of Animal Experiments of the Faculty of Veterinary Medicine, Cairo University.
Competing interests
The authors declare no competing interests.
Additional information
Handling Editor: Pierre Boudry
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Tawfeek, W.S., Kassab, A.S., Okasha, L.A. et al. The phenotypic and genetic characteristics of Pseudomonas anguilliseptica strains associated with mortalities in farmed sea bream and sea bass. Aquacult Int 32, 3973–3992 (2024). https://doi.org/10.1007/s10499-023-01360-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10499-023-01360-9