The Prevalence Rate, Pattern of Antibiotic Resistance, and Frequency of Virulence Factors of Pseudomonas aeruginosa Strains Isolated from Fish in Iran

Pseudomonas aeruginosa ( P. aeruginosa ) is a pathogenic bacterium and one of the seafood’s most common spoilage microorganisms. In this study, 470 ﬁsh samples collected randomly were evaluated for the presence of P. aeruginosa , antibiotic resistance, and frequency of virulence factors. Isolation of P. aeruginosa from ﬁsh samples was performed on cetrimide agar after an initial enrichment. Representative colonies were selected, and biochemical tests were conducted. An antibiotic resistance test was performed using the disk diﬀusion method. DNA was extracted, and antibiotic resistance genes, as well as virulence genes, were detected using PCR. Fresh ﬁsh showed the highest prevalence of P. aeruginosa (5%). No positive samples contaminated with P. aeruginosa were isolated from frozen ﬁsh samples. From smoked, salted, and dried ﬁsh, two samples (2.85–4%) were contaminated with P. aeruginosa . The antibiotic resistance against meropenem, imipenem, carbapenem, erythromycin, gentamicin, chloramphenicol, and enroﬂoxacin was 0%. The lowest antibiotic resistance pattern was observed in fresh ﬁsh, and the highest was observed in smoked, salted, and dried ﬁsh. Respectively, bla TEM , bla CTX-M , and bla SHV were the most abundant genes encoding antibiotic resistance. The most virulence genes were algD, algU, lasB, toxA, exoS, exoT , and apr . This study suggests that raw seafood could be a source of antibiotic-resistant P. aeruginosa and helps to spread resistance genes through the food chain. It seems that cross-contamination in the ﬁshing, transportation, and supply of seafood can cause increased contamination with Pseudomonas aeruginosa in these products. Therefore, the hygienic principles can eﬀectively reduce contamination by P. aeruginosa . Also, the prophylactic use of antibiotics in these products should be controlled.


Introduction
With the increasing consumption of fish and various types of seafood in Iran, the microbial quality of these products must be controlled. Fish is rich in protein, omega 3, omega 6, vitamins, and minerals. e presence of omega-3 fatty acids, including alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, has an influential role in consumer health and prevents some common diseases such as cardiovascular disease, Alzheimer's, stroke, asthma, cancer, and diabetes [1].
e Food and Agriculture Organization estimates the world consumption of fish as about 17.7 kg, and in some developed countries, its consumption has increased to 26 kg (Western Europe) and 80-90 kg (Japan). e FAO has announced the fish consumption in Iran as 7.7 kg [2].
Fish contains 80% water, 18% protein, 1% lipids, and 1% carbohydrates. Fish proteins contain large amounts of nonprotein nitrogen compounds (NPNs) and peptides. e pH of fish is generally higher than 6. In general, fish and seafood are more susceptible to microbial spoilage than other meat due to a lower pH and moisture content. e microbial spoilage may cause discoloration, off-odor, offflavor, and slime formation that makes it undesirable for consumption. e types of spoilage microorganisms in fish and seafood will depend on the type of product, muscle, and storage conditions [3].
Pseudomonas aeruginosa is one bacterium that can easily cause contamination and spoilage of fish meat.
is organism is a Gram-negative, aerobic, oxidase-positive, and catalase-positive bacillus, without spores. ey are found in various environments, have simple nutritional requirements, and survive under a wide range of temperatures. Pseudomonas aeruginosa with high pathogenicity has been causing the most common infections over 30 years [4]. is bacterium can be transmitted to humans by consuming contaminated meat products and causes gastrointestinal infections [5].
Pseudomonas aeruginosa is more critical than other spoilage bacteria due to its high resistance to a wide range of antibiotics. It has a wide range of virulence factors that can cause severe and aggressive infections in humans and animals. e genes encoding antibiotic resistance can be easily transmitted to humans through consuming contaminated seafood causing severe antibiotic resistance [6].
is Gram-negative bacterium is one of the factors limiting the shelf-life of fish and seafood and must be considered an important microbiological problem in seafood [7]. erefore, the present study aimed to investigate its prevalence in fish and types of fish products in Iran and the pattern of antibiotic resistance and virulence genes.

Sampling.
A total of 470 fish samples, including 200 fresh fish, 70 smoked fish, 70 salted fish, 50 dried fish, and 80 frozen fish samples, were collected randomly and under aseptic conditions from Isfahan, Ilam, Bushehr, and Mazandaran provinces (Iran) during six months. Samples were immediately sent to the laboratory.

Isolation of Pseudomonas aeruginosa from Fish Samples.
To isolate Pseudomonas aeruginosa from samples, 25 g of the sample was homogenized with 225 ml of peptone water. After initial enrichment, the bacteria were cultured in cetrimide agar (PCA) and incubated at 37°C for 24 hours. e colonies were selected based on color and odor (greenblue pigment with a specific odor). To confirm Pseudomonas aeruginosa species, biochemical tests such as fermentation of lactose, citrate, indole, oxidase, DNase, and hemolysis in a blood agar medium were performed. Colonies containing lactose-negative, citrate-positive, indole-negative, oxidasepositive, DNase-negative, and hemolytic bacteria were selected as Pseudomonas aeruginosa. ey were cultured in the brain heart infusion (BHI) medium at 37°C for 24 hours.
2.4. DNA Extraction. DNA was extracted from the overnight culture of bacteria in the brain heart infusion medium (Merck, Germany) using the DNA Purification Genomic Kit (Fermentas Germany). e DNA samples were then placed at −20°C until polymerase chain reaction (PCR).
e gene amplification process was done in 25 μl of a mixture including 1 unit of Taq DNA Polymerase (Fermentas, Lithuania), 200 μmol dNTP (Fermentas, Lithuania), 2.5 μl of 10x buffer solution (Fermentas, Lithuania), 1 μmol of manganese chloride (Fermentas, Lithuania) 10 picomoles of each primer, and 3 μl of template DNA, and nuclease-free water was added to 25 µl. e thermal program includes the initial denaturation at a temperature of 94°C for 6 minutes, denaturation stage at a temperature of 94°C for 60 seconds, annealing stage at a temperature of 55°C for 1 minute, extension stage at a temperature of 72°C for 5.5 minutes, and final extension stage at a temperature of 72°C for 5 minutes.

PCR Product Electrophoresis.
e distilled water was used as a negative control, and Pseudomonas aeruginosa strain ATCC 10145 was used as a positive control. PCR products were electrophoresed on 2% agarose gel. e gels were stained with Sybergreen (Fermentase, Germany), and DNA strips were evaluated using UV light.

Statistical Analysis.
e percentage of contamination in different sources and products has been calculated. e differences between groups were analyzed with SPSS statistical software (version 18) using the K 2 method. e significant level was determined at p < 0.05.

Isolation of Pseudomonas aeruginosa from Samples.
In this study, a total of 470 fish samples and fish products were evaluated for the presence of Pseudomonas aeruginosa.
e results of contaminated samples based on the culture and molecular method (PCR) are summarized in Table 2. As the results show, the prevalence rate in samples was reported at 14.7%. e fresh fish showed the highest prevalence rate of Pseudomonas aeruginosa. No positive samples with Pseudomonas aeruginosa were isolated from frozen fish samples. e smoked (2.85%), salted (2.85%), and dried fish (4%) samples were found to be contaminated by Pseudomonas aeruginosa. No significant difference was observed between the samples (p > 0.05).
Pseudomonas aeruginosa count in fish samples is significant because this bacterium is often considered an indicator of food quality and may cause food-borne illnesses [9,10]. e present study showed the highest and lowest prevalence of Pseudomonas aeruginosa in fresh and processed fish. Preservation processes such as smoking, salting, and drying may have played an essential role in reducing the bacterial population in these products.
Our results confirmed the contamination of fish samples with Pseudomonas aeruginosa. e results may be due to differences in hygienic principles during fishing, handling, freezing, storage, and thawing of fish, as Salem et al. reported [17]. Figures 1-4 summarize the antibiotic resistance of Pseudomonas aeruginosa strains isolated from fish and fish products. e results show that the prevalence of antibiotic resistance against meropenem, imipenem, carbapenem, erythromycin, gentamicin, chloramphenicol, and enrofloxacin was reported to be 0%. e prevalence of antibiotic resistance against antibiotics used in frozen fish was 0%. e highest antibiotic resistance was observed in smoked, salted, and dried fish samples. Pseudomonas aeruginosa was resistant to tetracycline, cefotaxime, chloramphenicol, imipenem, carbapenem, and penicillin antibiotics. Four semisensitive samples were reported against doxycycline, gentamicin, penicillin, and ampicillin antibiotics.

e Antibiotic Resistance Pattern of Pseudomonas aeruginosa Strains.
Pseudomonas aeruginosa strains isolated in Egypt showed multidrug resistance (MDR) to amoxicillin, cefotaxime, tetracycline, and gentamicin [11]. Carol et al. [19] observed that Pseudomonas aeruginosa had high resistance to amoxicillin, moderate resistance to ampicillin, ceftazidime, nitrofurantoin, and gentamicin, and sensitivity to tobramycin, cefotaxime, ethylene, ciprofloxacin, and amikacin.
Benie et al. [5] examined the prevalence of Pseudomonas aeruginosa in beef, fresh fish, and smoked fish. ey found that Pseudomonas aeruginosa strains were mainly resistant to cefepime, imipenem, ceftazidime, ciprofloxacin, and piperacillin. e resistance of Pseudomonas aeruginosa to cephalosporins (ceftazidime and cefepime) may be due to chromosomal mechanisms (disruption of the OprD purine) and an association of resistance mechanisms such as extended-spectrum beta-lactamase (ESBL).
In the study by Benie et al. [12], Pseudomonas aeruginosa isolates were resistant to aztreonam, ticarcillin, and ciprofloxacin. Aman et al. [20] found resistance of Pseudomonas aeruginosa to cefepime (34%) and ceftazidime (37%). Imipenem resistance is due to the reduction of purine permeability, according to Bricha et al. [21]. It is related to the enzymatic mechanism with carbapenemases, which causes a high level of resistance to all beta-lactamases.
Comparison of the results obtained in the present study with other studies shows that antibiotic resistance differs     Journal of Food Quality across countries. e antibiotic resistance of Pseudomonas aeruginosa seems to be constantly changing, and it is necessary to be attending to this change in different countries. Figure 1-4 summarize the antibiotic resistance and virulence genes of Pseudomonas aeruginosa isolated from fish samples. As can be seen, the most abundant genes encoding antibiotic resistance in Pseudomonas aeruginosa strains were bla TEM , bla CTX-M , and bla SHV from smoked, salted, and dried fish samples, respectively. e most abundant virulence genes detected in Pseudomonas aeruginosa were algD, algU, lasB, toxA, exoS, exoT, and apr.

e Virulence and Antibiotic Resistance Genes.
ese findings are consistent with those of other researchers [11,12]. Nonadherent virulence factors play an essential role in bacterial survival under specific conditions such as human blood, iron deficiency, and intestinal diseases. However, these nonadherent virulence factors are not essential for the pathogenesis of food-borne illness.
Algammal et al. [11] reported the bla CTXM , bla TEM , and tetA genes as the significant antibiotic resistance genes in Pseudomonas aeruginosa strains isolated from fish, which induces a pattern of resistance to cefotaxime, amoxicillin,    is highlights multidrug resistance (MDR) of Pseudomonas aeruginosa strains in public health. Benie et al. [12] identified bla SHV , bla TEM , bla PER , bla VEB , bla IMP , and bla VIM genes in Pseudomonas aeruginosa isolated from beef, fresh fish, and smoked fish.
Our findings differed from other studies mentioned above, which could be due to the geographical conditions and the level of health and improper use of some antibiotics in the livestock. Figures 5-10 show electrophoresis profiles of the genes detected in this study.

Conclusion
e results of this study confirmed the prevalence of Pseudomonas aeruginosa in fish and seafood, which is a severe warning to prevent the further spread of this microorganism. Due to the abundance of Pseudomonas aeruginosa in meat products, especially fish, much attention should be paid to the health and quality of meat products. Fish play an essential role in human nutrition. ey are a suitable food environment for the growth of bacteria such as Pseudomonas aeruginosa, so farms should be completely healthy.
Fish should be caught by public health standards and kept out of direct and indirect contact with external sources of contamination at all stages of fishing, collection, handling, and transportation. erefore, with the lack of hygienic and cooling systems, Pseudomonas aeruginosa in the fish will increase. e hygienic principles must be applied to reduce contamination with Pseudomonas aeruginosa.
Data Availability e data that support the findings of this study are available on request from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.