# Prevalence, Antimicrobial Resistance, and Molecular Description ofPseudomonas aeruginosa Isolated from Meat and Meat Products

Authors: Manizhe Rezaloo; Abbasali Motalebi; Zohreh Mashak; Amirali Anvar
Journal: Journal of Food Quality
 (2022)

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## Abstract

Resistant and virulentPseudomonas aeruginosa (P. aeruginosa) bacteria are measured as the major cause of food spoilage and food-borne diseases. This survey assesses the prevalence, antibiotic resistance properties, and virulence factors distribution in P. aeruginosa bacteria isolated from meat and meat products. A total of 370 raw, frozen, and imported bovine meat samples and diverse types of meat product samples were collected from Alborz province, Iran. P. aeruginosa bacteria were identified by culture. Disk diffusion was used to assess the antibiotic resistance of bacteria. Furthermore, the PCR was used to assess the virulence and antibiotic resistance genes. Twenty nine out of 370 (7.83%) samples were contaminated with P. aeruginosa. Imported frozen bovine meat (20%) harbored the highest distribution, while sausage (2%) harbored the lowest. High resistance rates were observed toward ampicillin (89.65%), penicillin (86.20%), tetracycline (82.75%), cefoxitin (37.93%), gentamicin (34.48%), and clindamycin (31.03%). The most commonly detected antibiotic resistance genes were blaDHA (93.10%), blaCTX-M (83.65%), and blaSHV (48.27%). BlaDHA (93.10%), blaCTX-M (83.65%), and blaSHV (48.27%) were the most frequently detected resistance genes. The most commonly detected virulence genes were exoS (75.86%), lasA (68.96%), exoU (58.62%), lasB (51.72%), plcH (48.27%), and algD (44.82%). Meat and meat product samples may be sources of P. aeruginosa, which show an important threat to their consumption. Nevertheless, additional inquiries are obligatory to find supplementary epidemiological properties of P. aeruginosa in meat and meat product samples.


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## Body

## 1. Introduction

Meat is one of the most important sources of protein in the world. Additionally, red meat is a rich source of fatty acids, vitamins, proteins, and minerals. Red meat also can be converted into a wide range of meat products, such as burgers, kebabs, sausages, and salami. Reports indicate that meat products are a large part of the world’s diet [1, 2]. However, there are many reports of food-borne illnesses due to consuming contaminated meat and meat products, which increases the importance of hygiene and inspection of these products[3–6].Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic Gram-negative bacterium responsible for hospital infections, particularly bacteremia, urinary and respiratory tract infections (UTIs and RTIs, respectively), soft and hard tissues, and gastrointestinal and systemic infections [7]. It is also responsible for severe burn and wound infections in immunosuppressed patients [8]. The bacterium may be involved in food spoilage characterized by off-flavors, pigment secretion, and slime and malodor production [9]. Food spoilage caused by this bacterium is a thoughtful global issue, particularly in developing countries, owing to insufficient processing and refrigeration technologies.There is a growing sign of the occurrence of food-borne infections caused byP. aeruginosa [10]. Diverse virulence factors are responsible for the occurrence of infections caused by this bacterium. Phenazine operons (phzH, phzM, and phzS) secrete the precursor proteins that encode three phenazine compounds [11]. These compounds are responsible for increasing intracellular oxidative effects [11]. Exoenzymes (exoS, exoT, exoU, and exoY), alkaline protease (apr), toxin A (toxA), elastase gene A and B (lasA and lasB), alginate-encoded genes (algD and algU), hemolytic and nonhemolytic phospholipase C (plcH and plcN), pilus genes (pilA and pilB), and pyoverdine encoded gene (pvd) are other P. aeruginosa virulence factors [12, 13]. The role of these factors is often adhesions and attachments, inflammatory reactions, and ultimately invasion of the host cell.Another critical aspect ofP. aeruginosa-related infections is antibiotic resistance. P. aeruginosa progresses resistance to varied types of antibiotic agents [14]. In 2017, multidrug-resistant P. aeruginosa was responsible for about 32,500 infections and 2,700 deaths in hospitalized patients in the United States [15]. Resistant strains cause more complicated diseases for an extended period, which causes a lot of economic losses due to treatment and control. P. aeruginosa reveals resistance to varied types of antimicrobial agents, predominantly β-lactams, aminoglycosides, tetracyclines, quinolones, and macrolides [16]. Throughout the last years, extended-spectrum beta-lactamase (ESBLs) types (encoding resistance to all beta-lactams, except carbapenems) have arisen in the hospital and community setting among P. aeruginosa strains on plasmids commonly harboring other genes encoding resistance to non-beta-lactam antibiotics. The most important and clinically relevant genes encoding ESBL enzymes in P. aeruginosa are blaSHV, blaTEM, blaDHA, blaOXA, blaVEB, and blaCTX-M genes [17, 18]. Additionally, quinolone (nalidixic acid, ciprofloxacin, ofloxacin, etc.) resistance has been increasingly reported in P. aeruginosa clinical isolates. This resistance is mainly associated to point mutations in gyrA (encoding GyrA subunit of DNA gyrase) and parC (encoding parC subunit of topoisomerase IV) [19].Rendering the boost portion of meat consumption in Iran and alsoP. aeruginosa’s importance as a food spoilage agent, the present survey was performed to assess the prevalence as well as phenotypic and genotypic patterns of antibiotic resistance and virulence factors distribution in P. aeruginosa bacteria isolated from diverse kinds of meat and meat products.


## 2. Materials and Methods

### 2.1. Ethics

This research was confirmed by the Ethics Committee of the College of Veterinary Medicine, Islamic Azad University.


### 2.2. Study Design

The targeted population of the present study was fresh meat, frozen meat, imported frozen meat, hamburgers, kebabs, sausages, and salami samples presented in the winter of 2020 in Alborz province, Iran. Samples were collected from different sales centers in Alborz province. The sampling unit for each sample was 100 g of meat or meat products.


### 2.3. Sampling Procedure

A total of 370 dissimilar kinds of meat and meat product samples, such as fresh bovine meat (n = 60), frozen bovine meat (n = 60), imported frozen bovine meat (n = 50), burger (n = 50), kebab (n = 50), sausage (n = 50), and salami (n = 50), were randomly collected from the sale centers of the Alborz province, Iran. The samples (100 g) were transferred to Food Hygiene Research Center using cool boxes.


### 2.4. Bacterial Isolation and Identification

Twenty-five grams of the collected meat and meat product samples were put in sterile stomacher bags containing 225 ml peptone water (Oxoid, Basingstoke, UK). The bags underwent maceration within the stomacher (Seward 400 circulator, 4 min at 260 beats per min). A total of 100μl homogenate samples were placed on CN selective agar (Oxoid SR 102E, UK) supplemented with Pseudomonas agar base (Oxoid, UK). Media were incubated at 37°C for 24 h in aerobic conditions. Microscopic morphology, Gram-staining, oxidase, urease, and catalase activities, starch and casein hydrolysis, methyl red and Voges Proskauer (MR-VP) and gelatin liquefaction and indole and citrate utilization tests were applied to identify that P. aeruginosa was identified by API 20NE strips (BioMeriouxVitek, Inc., MO, USA) system. The polymerase chain reaction (PCR) was used to identify P. aeruginosa isolates based on the detection of rpoB household genes [20].


### 2.5. Antimicrobial Susceptibility Testing

A simple disk diffusion method (Kirby Bauer) was used to investigate the pattern of antibiotic resistance.P. aeruginosa isolates were cultured overnight in Tryptone Soya broth (TSB, Merck, Germany) medium and incubated in aerobic conditions at 37°C for 24 h. After the preparation of 0.5 McFarland dilution, isolates were cultured on the Mueller–Hinton agar (Merck, Germany) in the presence of diverse antibiotic discs. The following antibiotics were used (µg/disk): tetracycline (30), chloramphenicol (30), sulfamethoxazole (25), gentamicin (10), ciprofloxacin (5), trimethoprim (5), ampicillin (10), penicillin (10), cefoxitin (10); clindamycin (2), imipenem (10), and aztreonam (30) (Oxoid, UK). Cultures were incubated at 37°C for 24 h. To assess the resistance pattern, the diameter of the growth inhibition halo was measured. In this experiment, P. aeruginosa ATCC 1014 was used as a positive control. Findings were interpreted according to the Antimicrobial Susceptibility Testing European Committee (EUCAST, 2018) [21] and CLSI criteria (2016 and 2018) [22–24].


### 2.6. Detection of Virulence and Antibiotic Resistance Genes

P. aeruginosa isolates were subcultured on TSB media (48 h at 37°C). Isolate’s DNA was extracted using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Guidelines of the kit instruction were applied. Quality (using electrophoresis on a 2% agarose gel) and quantity (using a spectrophotometer (A260/A280)) of extracted DNA were measured [25]. Table 1 indicates the PCR ingredients and circumstances applied for the virulence and antibiotic resistance genes identification [26–29].Table 1
PCR conditions [26–30].
Molecular charactersTargeted genesPrimer sequence (5′-3′)PCR product (bp)PCR programsPCR volume (50μL)Virulence factorsalgDF: AAG GCG GAA ATG CCA TCT CC2751 cycle:2 min at 95°C30 cycles:30 s at 94°C30 s at 58°C60 s at 72°C1 cycle:7 min at 72°CdNTP: 200μMPCR buffer (10X): 5 μLPrimer F: 0.5 μMPrimer R: 0.5 μMMgcl2: 1.5 mMDNA: 2.5 μLTaq DNA polymerase: 1.25 UR: AGG GAA GTT CCG GGC GTT TGalgUF: CGC GAA CCG CAC CAT CGC TC410R: GCC GCA CGT CAC GAG CexoT∗F: CAA TCA TCT CAG CAG AAC CC11591 cycle:5 min at 96°C30 cycles:30 s at 94°C30 s at 47–63 0C1 min at 72°C1 cycle:3 min at 72°CR: TGT CGT AGA GGA TCT CCT GlasAF: GCA GCA CAA AAG ATC CC1075R: GAA ATG CAG GTG CGG TCpvdAF: GAC TCA GGC AAC TGC AAC1281R: TTC AGG TGC TGG TAC AGGlasBF: ACA CAA TAC ATA TCA ACT TCG C2841 cycle:3 min at 94°C30 cycles:30 s at 94°C60 s at 55°C90 s at 72°C1 cycle:5 min at 72°CR: AGT GTG TTT AGA ATG GTG ATCtoxAF: ATG TGC AGY ACC AGT AAR GT270R: TGG GTR AAR TAR GTS ACC AGAplcHF: CAC ACG GAA GGT TAA TTC TGA608R: CGG TTA RAC GGC TGA ACC TGplcNF: CGA CTT CCA TTT CCC GAT GC481R: GGA CTC TGC AAC AAA TAC GCexoSF: GTG TGC TTT ATG CCA TGA G444R: GGT TTC CTT TTC CAG GTCexoYF: TAT CGA CGG TCA TCG TCA GGT10351 cycle:5 min at 96°C30 cycles:30 s at 94°C30 s at 47–63 0C1 min at 72°C1 cycle:3 min at 72°CR: TTG ATG CAC TCG ACC AGC AAGexoUF: GAT TCC ATC ACA GGC TCG3308R: CTA GCA ATG GCA CTA ATC GaprF: TGT CCA GCA ATT CTC TTG CR: CGT TTT CCA CGG TGA CC1017phzMF: ATG GAG AGC GGG ATC GAC AG875R: ATG CGG GTT TCC ATC GGC AGphzSF: TCG CCA TGA CCG ATA CGC TC1752R: ACA ACC TGA GCC AGC CTT CCphzHF: GCC AAG GTT TGT TGT CGG1036R: CGC ATT GAC GAT ATG GAA CpilAF: ACA GCA TCC AAC TGA GCG1675R: TTG ACT TCC TCC AGG CTGpilBF: TCG AAC TGA TGA TCG TGG408R: CTT TCG GAG TGA ACA TCGAntibiotic resistance genesblaTEMaF: ATG AGT ATT CAA CAT TTC CG7471 cycle:5 min at 96°C35 cycles:60 s at 96°C60 s at 58°C60 s at 72°C1 cycle:10 min at 72°CR: CTG ACA GTT ACC AAT GCT TAblaSHVbF: GGT TAT GCG TTA TAT TCG CC8671 cycle:5 min at 96°C35 cycles:60 s at 96°C60 s at 60°C60 s at 72°C1 cycle:10 min at 72°CR: TTA GCG TTG CCA GTG CTCblaOXAcF: ACA CAA TAC ATA TCA ACT TCG C885R: AGT GTG TTT AGA ATG GTG ATCblaCTX-MdF: ATG TGC AGY ACC AGT AAR GT5931 cycle:7 min at 94°C35 cycles:50 s at 94°C40 s at 50 0C60 s: At 72°C1 cycle:5 min at 72°CR: TGG GTR AAR TAR GTS ACC AGAblaDHAeF: CAC ACG GAA GGT TAA TTC TGA9701 cycle:5 min at 94°C35 cycles:30 s at 94°C45 s at 50°C60 s at 72°C1 cycle:8 min at 72°CR: CGG TTA RAC GGC TGA ACC TGblaVEBfF: CGA CTT CCA TTT CCC GAT GC10141 cycle:5 min at 96°C30 cycles:60 s at 96°C60 s at 55°C2 min at 72°C1 cycle:10 min at 72°CR: GGA CTC TGC AAC AAA TAC GCgyrAF: GTG TGC TTT ATG CCA TGA G2871 cycle:14 min at 95°C35 cycles:45 s at 95°C45 s at 51°C60 s at 71°C1 cycle:7 min at 71°CR: GGT TTC CTT TTC CAG GTCparCF: CAT CGT CTA CGC CAT GAG267R: AGC AGC ACC TCG GAA TAG∗Annealing temperatures of exoT, exoY, exoU, apr, phzM, phzS, phzH, lasA, lasB, pvdA, pilA, and pilB virulence factors were 54, 61.8, 61.6, 51, 54, 63, 51, 47, 57, 59, 59, and 54°C, respectively. aTemoneira: the name of the first patient providing the first sample. bSulfhydryl reagent variable. cActive on oxacillin. dActive on cefotaxime, first isolated at Munich. eDiscovered at Dhahran, Saudi Arabia. fVietnam extended-spectrum β-lactamase.PCR was done using a thermocycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany). For electrophoresis, 2.5% agarose gel containing ethidium bromide (0.1%, 0.4μg/ml) at a constant level of 120 V/208 mA was used. Visualization was done by UVI doc device (Grade GB004, Jencons PLC, London, UK).


### 2.7. Data Analysis

SPSS software (21.0 version, Chicago, IL, USA) was used for analysis. For data assessment, Chi-square and Fisher’s exact two-tailed tests were used.P value <0.05 was determined as levels of significance.


## 2.1. Ethics

This research was confirmed by the Ethics Committee of the College of Veterinary Medicine, Islamic Azad University.


## 2.2. Study Design

The targeted population of the present study was fresh meat, frozen meat, imported frozen meat, hamburgers, kebabs, sausages, and salami samples presented in the winter of 2020 in Alborz province, Iran. Samples were collected from different sales centers in Alborz province. The sampling unit for each sample was 100 g of meat or meat products.


## 2.3. Sampling Procedure

A total of 370 dissimilar kinds of meat and meat product samples, such as fresh bovine meat (n = 60), frozen bovine meat (n = 60), imported frozen bovine meat (n = 50), burger (n = 50), kebab (n = 50), sausage (n = 50), and salami (n = 50), were randomly collected from the sale centers of the Alborz province, Iran. The samples (100 g) were transferred to Food Hygiene Research Center using cool boxes.


## 2.4. Bacterial Isolation and Identification

Twenty-five grams of the collected meat and meat product samples were put in sterile stomacher bags containing 225 ml peptone water (Oxoid, Basingstoke, UK). The bags underwent maceration within the stomacher (Seward 400 circulator, 4 min at 260 beats per min). A total of 100μl homogenate samples were placed on CN selective agar (Oxoid SR 102E, UK) supplemented with Pseudomonas agar base (Oxoid, UK). Media were incubated at 37°C for 24 h in aerobic conditions. Microscopic morphology, Gram-staining, oxidase, urease, and catalase activities, starch and casein hydrolysis, methyl red and Voges Proskauer (MR-VP) and gelatin liquefaction and indole and citrate utilization tests were applied to identify that P. aeruginosa was identified by API 20NE strips (BioMeriouxVitek, Inc., MO, USA) system. The polymerase chain reaction (PCR) was used to identify P. aeruginosa isolates based on the detection of rpoB household genes [20].


## 2.5. Antimicrobial Susceptibility Testing

A simple disk diffusion method (Kirby Bauer) was used to investigate the pattern of antibiotic resistance.P. aeruginosa isolates were cultured overnight in Tryptone Soya broth (TSB, Merck, Germany) medium and incubated in aerobic conditions at 37°C for 24 h. After the preparation of 0.5 McFarland dilution, isolates were cultured on the Mueller–Hinton agar (Merck, Germany) in the presence of diverse antibiotic discs. The following antibiotics were used (µg/disk): tetracycline (30), chloramphenicol (30), sulfamethoxazole (25), gentamicin (10), ciprofloxacin (5), trimethoprim (5), ampicillin (10), penicillin (10), cefoxitin (10); clindamycin (2), imipenem (10), and aztreonam (30) (Oxoid, UK). Cultures were incubated at 37°C for 24 h. To assess the resistance pattern, the diameter of the growth inhibition halo was measured. In this experiment, P. aeruginosa ATCC 1014 was used as a positive control. Findings were interpreted according to the Antimicrobial Susceptibility Testing European Committee (EUCAST, 2018) [21] and CLSI criteria (2016 and 2018) [22–24].


## 2.6. Detection of Virulence and Antibiotic Resistance Genes

P. aeruginosa isolates were subcultured on TSB media (48 h at 37°C). Isolate’s DNA was extracted using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Guidelines of the kit instruction were applied. Quality (using electrophoresis on a 2% agarose gel) and quantity (using a spectrophotometer (A260/A280)) of extracted DNA were measured [25]. Table 1 indicates the PCR ingredients and circumstances applied for the virulence and antibiotic resistance genes identification [26–29].Table 1
PCR conditions [26–30].
Molecular charactersTargeted genesPrimer sequence (5′-3′)PCR product (bp)PCR programsPCR volume (50μL)Virulence factorsalgDF: AAG GCG GAA ATG CCA TCT CC2751 cycle:2 min at 95°C30 cycles:30 s at 94°C30 s at 58°C60 s at 72°C1 cycle:7 min at 72°CdNTP: 200μMPCR buffer (10X): 5 μLPrimer F: 0.5 μMPrimer R: 0.5 μMMgcl2: 1.5 mMDNA: 2.5 μLTaq DNA polymerase: 1.25 UR: AGG GAA GTT CCG GGC GTT TGalgUF: CGC GAA CCG CAC CAT CGC TC410R: GCC GCA CGT CAC GAG CexoT∗F: CAA TCA TCT CAG CAG AAC CC11591 cycle:5 min at 96°C30 cycles:30 s at 94°C30 s at 47–63 0C1 min at 72°C1 cycle:3 min at 72°CR: TGT CGT AGA GGA TCT CCT GlasAF: GCA GCA CAA AAG ATC CC1075R: GAA ATG CAG GTG CGG TCpvdAF: GAC TCA GGC AAC TGC AAC1281R: TTC AGG TGC TGG TAC AGGlasBF: ACA CAA TAC ATA TCA ACT TCG C2841 cycle:3 min at 94°C30 cycles:30 s at 94°C60 s at 55°C90 s at 72°C1 cycle:5 min at 72°CR: AGT GTG TTT AGA ATG GTG ATCtoxAF: ATG TGC AGY ACC AGT AAR GT270R: TGG GTR AAR TAR GTS ACC AGAplcHF: CAC ACG GAA GGT TAA TTC TGA608R: CGG TTA RAC GGC TGA ACC TGplcNF: CGA CTT CCA TTT CCC GAT GC481R: GGA CTC TGC AAC AAA TAC GCexoSF: GTG TGC TTT ATG CCA TGA G444R: GGT TTC CTT TTC CAG GTCexoYF: TAT CGA CGG TCA TCG TCA GGT10351 cycle:5 min at 96°C30 cycles:30 s at 94°C30 s at 47–63 0C1 min at 72°C1 cycle:3 min at 72°CR: TTG ATG CAC TCG ACC AGC AAGexoUF: GAT TCC ATC ACA GGC TCG3308R: CTA GCA ATG GCA CTA ATC GaprF: TGT CCA GCA ATT CTC TTG CR: CGT TTT CCA CGG TGA CC1017phzMF: ATG GAG AGC GGG ATC GAC AG875R: ATG CGG GTT TCC ATC GGC AGphzSF: TCG CCA TGA CCG ATA CGC TC1752R: ACA ACC TGA GCC AGC CTT CCphzHF: GCC AAG GTT TGT TGT CGG1036R: CGC ATT GAC GAT ATG GAA CpilAF: ACA GCA TCC AAC TGA GCG1675R: TTG ACT TCC TCC AGG CTGpilBF: TCG AAC TGA TGA TCG TGG408R: CTT TCG GAG TGA ACA TCGAntibiotic resistance genesblaTEMaF: ATG AGT ATT CAA CAT TTC CG7471 cycle:5 min at 96°C35 cycles:60 s at 96°C60 s at 58°C60 s at 72°C1 cycle:10 min at 72°CR: CTG ACA GTT ACC AAT GCT TAblaSHVbF: GGT TAT GCG TTA TAT TCG CC8671 cycle:5 min at 96°C35 cycles:60 s at 96°C60 s at 60°C60 s at 72°C1 cycle:10 min at 72°CR: TTA GCG TTG CCA GTG CTCblaOXAcF: ACA CAA TAC ATA TCA ACT TCG C885R: AGT GTG TTT AGA ATG GTG ATCblaCTX-MdF: ATG TGC AGY ACC AGT AAR GT5931 cycle:7 min at 94°C35 cycles:50 s at 94°C40 s at 50 0C60 s: At 72°C1 cycle:5 min at 72°CR: TGG GTR AAR TAR GTS ACC AGAblaDHAeF: CAC ACG GAA GGT TAA TTC TGA9701 cycle:5 min at 94°C35 cycles:30 s at 94°C45 s at 50°C60 s at 72°C1 cycle:8 min at 72°CR: CGG TTA RAC GGC TGA ACC TGblaVEBfF: CGA CTT CCA TTT CCC GAT GC10141 cycle:5 min at 96°C30 cycles:60 s at 96°C60 s at 55°C2 min at 72°C1 cycle:10 min at 72°CR: GGA CTC TGC AAC AAA TAC GCgyrAF: GTG TGC TTT ATG CCA TGA G2871 cycle:14 min at 95°C35 cycles:45 s at 95°C45 s at 51°C60 s at 71°C1 cycle:7 min at 71°CR: GGT TTC CTT TTC CAG GTCparCF: CAT CGT CTA CGC CAT GAG267R: AGC AGC ACC TCG GAA TAG∗Annealing temperatures of exoT, exoY, exoU, apr, phzM, phzS, phzH, lasA, lasB, pvdA, pilA, and pilB virulence factors were 54, 61.8, 61.6, 51, 54, 63, 51, 47, 57, 59, 59, and 54°C, respectively. aTemoneira: the name of the first patient providing the first sample. bSulfhydryl reagent variable. cActive on oxacillin. dActive on cefotaxime, first isolated at Munich. eDiscovered at Dhahran, Saudi Arabia. fVietnam extended-spectrum β-lactamase.PCR was done using a thermocycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany). For electrophoresis, 2.5% agarose gel containing ethidium bromide (0.1%, 0.4μg/ml) at a constant level of 120 V/208 mA was used. Visualization was done by UVI doc device (Grade GB004, Jencons PLC, London, UK).


## 2.7. Data Analysis

SPSS software (21.0 version, Chicago, IL, USA) was used for analysis. For data assessment, Chi-square and Fisher’s exact two-tailed tests were used.P value <0.05 was determined as levels of significance.


## 3. Results

### 3.1.P. aeruginosa Prevalence

TheP. aeruginosa prevalence in different examined meat and meat product samples is shown in Table 2. Twenty nine of 370 (7.83%) meat and meat product samples were contaminated with P. aeruginosa. Imported frozen bovine meat (20.00%) harbored the highest prevalence of bacteria, while sausage (2.00%) harbored the lowest. Data analysis revealed significant differences between the types of samples and the prevalence of P. aeruginosa (P<0.05). Statistically, significant differences were observed for the P. aeruginosa prevalence between fresh and frozen bovine meat samples (P<0.05), fresh and imported bovine meat samples (P<0.05), and meat and meat product samples (P<0.05). The applied method failed to detect any P. aeruginosa in sausage samples.Table 2
P. aeruginosa prevalence amongst the examined meat and meat product samples.
Sample typesN. collectedN. positive forP. aeruginosa (%)Fresh bovine meat603 (5.00)Frozen bovine meat608 (13.33)Imported frozen bovine meat5010 (20.00)Meat productsBurger503 (6.00)Kebab504 (8.00)Sausage501 (2.00)Salami500Total2008 (4.00)Total37029 (7.83)


### 3.2.P. aeruginosa Antibiotic Resistance Pattern

P. aeruginosa antibiotic resistance pattern is shown in Table 3. P. aeruginosa bacteria showed the highest resistance rate toward ampicillin (89.65%), penicillin (86.20%), and tetracycline (82.75%) antibiotic agents. The resistance rates of examined bacteria toward cefoxitin, gentamicin, clindamycin, and sulfamethoxazole were 37.93%, 34.48%, 31.03%, and 27.58%, respectively. The lowest resistance rate was observed against chloramphenicol (17.24%) and imipenem (20.68%). P. aeruginosa isolates of imported frozen bovine meat samples harbored the highest resistance against examined antibiotic agents (P<0.05). Data analysis revealed significant differences between the types of samples and the resistance rate of bacteria against antibiotic agents (P<0.05).Table 3
P. aeruginosa antibiotic resistance pattern.
Samples (N.P. aeruginosa)N. isolates resist to each antibiotic agent (%)TCSGCIPTAPCefCLNImAZFresh bovine meat (3)2 (66.66)01 (33.33)1 (33.33)1 (33.33)1 (33.33)3 (100)2 (66.66)1 (33.33)1 (33.33)01 (33.33)Frozen bovine meat (8)7 (87.50)02 (25)3 (37.50)2 (25)1 (12.50)8 (100)8 (100)2 (25)2 (25)1 (12.50)2 (25)Imported frozen bovine meat (10)10 (100)2 (20)3 (30)7 (70)5 (50)3 (30)10 (100)10 (100)5 (50)4 (40)3 (30)3 (30)Meat productsBurger (3)2 (66.66)1 (33.33)1 (33.33)2 (66.66)1 (33.33)02 (66.66)2 (66.66)1 (33.33)01 (33.33)0Kebab (4)2 (50)1 (25)1 (25)1 (25)1 (25)2 (66.66)2 (50)2 (50)1 (25)1 (25)1 (25)1 (25)Sausage (1)1 (100)1 (100)01 (100)001 (100)1 (100)1 (100)1 (100)00Total (8)5 (62.50)3 (37.50)2 (25)4 (50)2 (25)2 (12.50)5 (62.50)5 (62.50)3 (37.50)2 (12.50)2 (12.50)1 (12.50)Total (29)24 (82.75)5 (17.24)8 (27.58)15 (51.72)10 (34.48)7 (24.13)26 (89.65)25 (86.20)11 (37.93)9 (31.03)6 (20.68)7 (24.13)∗T: tetracycline (30 μg/disk); C: chloramphenicol (30 μg/disk); S: sulfamethoxazole (25 μg/disk); G: gentamicin (10 μg/disk); CIP: ciprofloxacin (5 μg/disk); T: trimethoprim (5 μg/disk); A: ampicillin (10 μg/disk); P: penicillin (10 μg/disk); Cef: cefoxitin (10 μg/disk); CLN: clindamycin (2 μg/disk); Im: imipenem (10 μg/disk); AZ: aztreonam (30 μg/disk).


### 3.3.P. aeruginosa Antibiotic Resistance Genes

Gel electrophoresis of the PCR products of virulence factors and antibiotic resistance genes is presented in Figure1. The distribution of P. aeruginosa antibiotic resistance genes is shown in Table 4. According to obtained data, blaDHA (93.10%), blaCTX-M (83.65%), and blaSHV (48.27%) were the most frequently detected antibiotic resistance genes amongst the P. aeruginosa isolates. Findings showed that blaTEM (20.68%) and gyrA (24.13%) had lower frequencies amongst the examined antibiotic resistance genes. The distribution of blaOXA and blaVEB was 34.48% and 34.48%, respectively. P. aeruginosa isolates of imported frozen bovine meat samples harbored a higher distribution of antibiotic resistance genes (P<0.05). Data analysis revealed significant differences between the types of samples and the distribution of antibiotic-resistance genes (P<0.05).Figure 1
(a) Gel electrophoresis of PCR products for the detection ofalgD and algU virulence factors. M 100 bp ladder, 1–3: positive samples for algD (275 bp) and algU (410 bp) virulence factors, PC: positive control, and NC: negative control. (b) Gel electrophoresis of PCR products for the detection of lasB, toxA, plcH, plcN, and exoS virulence factors. M 100 bp ladder, 1–9: positive samples for virulence factors and their positive controls, and NC: negative control. (c) Gel electrophoresis of PCR products for the detection of pilA, pilB, phzM, apr, phzH, phzS, exoU, and exoY virulence factors. M 1000 bp ladder, 1–8 and 10–14 and 16: positive samples for virulence factors and their positive controls, NC: negative control, and 9 and 15: negative samples for virulence factors. (d) Gel electrophoresis of PCR products for the detection of lasA, exoT, and pvdA virulence factors. M 1000 bp ladder, 1–6: positive samples for virulence factors and their positive controls, NC: negative control. (e) Gel electrophoresis of PCR products for the detection of blaTEM, blaOXA, and blaSHV antibiotic resistance genes. M 100 bp ladder, 1–6: positive samples for virulence factors and their positive controls, NC: negative control. (f) Gel electrophoresis of PCR products for the detection of the blaCTX-M antibiotic resistance gene. M 100 bp ladder, 1–3: positive samples, PC: positive control, and NC: negative control. (g) Gel electrophoresis of PCR products for the detection of blaDHA antibiotic resistance gene. M 100 bp ladder, 1–3: positive samples, PC: positive control, and NC: negative control. (h) Gel electrophoresis of PCR products for the detection of blaVEB antibiotic resistance gene. M 100 bp ladder, 1–5: positive samples, PC: positive control, and NC: negative control. (i) Gel electrophoresis of PCR products for the detection of gyrA and parC antibiotic resistance gene. M 100 bp ladder, 1–4: positive samples, PC: positive control, and NC: negative control.Table 4
P. aeruginosa antibiotic resistance genes.
Samples (N.P. aeruginosa)N. isolates harbored each antibiotic resistance gene (%)blaTEMblaSHVblaOXAblaCTX-MblaDHAblaVEBgyrAparCFresh bovine meat (3)1 (33.33)1 (33.33)1 (33.33)2 (66.66)2 (66.66)1 (33.33)1 (33.33)1 (33.33)Frozen bovine meat (8)1 (12.50)2 (25)2 (25)7 (87.50)8 (100)3 (37.50)2 (25)1 (12.50)Imported frozen bovine meat (10)3 (30)7 (70)4 (40)10 (100)10 (100)3 (30)2 (20)2 (20)Meat productsBurger (3)1 (33.33)1 (33.33)1 (33.33)2 (66.66)3 (100)1 (33.33)1 (33.33)0Kebab (4)1 (25)2 (50)2 (50)4 (100)3 (75)2 (50)1 (25)1 (25)Sausage (1)01 (100)01 (100)1 (100)000Total (8)2 (12.50)4 (50)3 (37.50)7 (87.50)7 (87.50)3 (37.50)2 (12.50)1 (12.50)Total (29)6 (20.68)14 (48.27)10 (34.48)26 (89.65)27 (93.10)10 (34.48)7 (24.13)5 (17.24)


### 3.4.P. aeruginosa Virulence Factors

The distribution ofP. aeruginosa virulence factors amongst the examined meat and meat product samples is shown in Table 5. Findings showed that exoS (75.86%), lasA (68.96%), exoU (58.62%), lasB (51.72%), plcH (48.27%), and algD (44.82%) were the most frequently detected virulence factors. Distribution of pilA, phzM, toxA, and exoT virulence factors was 24.13%, 20.68%, 17.24%, and 17.24%, respectively. PhzH (3.44%), plcN (6.89%), pvdA (6.89%), and pilB (6.89%) had the lower distribution among examined virulence factors. Statistically, significant differences were observed amid the distribution of algD and algU (P<0.05), plcH and plcN (P<0.05), exoS and exoT (P<0.05), exoU and exoT (P<0.05), exoS and exoY (P<0.05), exoU and exoY (P<0.05), phzM and phzH (P<0.05), lasA and lasB (P<0.05), and pilA and pilB (P<0.05). P. aeruginosa isolates of imported frozen bovine meat samples harbored a higher distribution of virulence factors (P<0.05). Data analysis revealed significant differences between the types of samples and the distribution of virulence factors (P<0.05).Table 5
P. aeruginosa virulence factors.
Samples (N.P. aeruginosa)N. isolates resist each virulence factor (%)algDalgUtoxAplcHplcNexoSexoTexoYexoUaprphzMphzSphzHlasAlasBpvdApilApilBFresh bovine meat (3)1 (33.33)01 (33.33)2 (66.66)02 (66.66)1 (33.33)02 (66.66)00002 (66.66)2 (66.66)01 (33.33)0Frozen bovine meat (8)3 (37.50)1 (12.50)1 (12.50)3 (37.50)06 (75)1 (12.50)04 (50)1 (12.50)1 (12.50)1 (12.50)05 (62.50)3 (37.50)01 (12.50)0Imported frozen bovine meat (10)5 (50)2 (20)2 (20)6 (60)2 (20)8 (80)2 (20)2 (20)7 (70)2 (20)3 (30)1 (10)1 (10)8 (80)6 (60)1 (12.50)4 (40)1 (12.50)Meat productsBurger (3)1 (33.33)001 (33.33)02 (66.66)1 (33.33)01 (33.33)01 (33.33)002 (66.66)1 (33.33)001 (33.33)Kebab (4)2 (50)1 (25)1 (25)1 (25)03 (75)01 (25)2 (50)1 (25)1 (25)1 (25)02 (50)2 (50)1 (25)1 (25)0Sausage (1)1 (100)001 (100)01 (100)001 (100)00001 (100)1 (100)000Total (8)4 (50)1 (12.50)1 (12.50)3 (37.50)05 (62.50)1 (12.50)1 (12.50)4 (50)1 (12.50)2 (25)1 (12.50)05 (62.50)4 (50)1 (12.50)1 (12.50)1 (12.50)Total (29)13 (44.82)4 (13.79)5 (17.24)14 (48.27)2 (6.89)22 (75.86)5 (17.24)3 (10.34)17 (58.62)4 (13.79)6 (20.68)3 (10.34)1 (3.44)20 (68.96)15 (51.72)2 (6.89)7 (24.13)2 (6.89)


## 3.1.P. aeruginosa Prevalence

TheP. aeruginosa prevalence in different examined meat and meat product samples is shown in Table 2. Twenty nine of 370 (7.83%) meat and meat product samples were contaminated with P. aeruginosa. Imported frozen bovine meat (20.00%) harbored the highest prevalence of bacteria, while sausage (2.00%) harbored the lowest. Data analysis revealed significant differences between the types of samples and the prevalence of P. aeruginosa (P<0.05). Statistically, significant differences were observed for the P. aeruginosa prevalence between fresh and frozen bovine meat samples (P<0.05), fresh and imported bovine meat samples (P<0.05), and meat and meat product samples (P<0.05). The applied method failed to detect any P. aeruginosa in sausage samples.Table 2
P. aeruginosa prevalence amongst the examined meat and meat product samples.
Sample typesN. collectedN. positive forP. aeruginosa (%)Fresh bovine meat603 (5.00)Frozen bovine meat608 (13.33)Imported frozen bovine meat5010 (20.00)Meat productsBurger503 (6.00)Kebab504 (8.00)Sausage501 (2.00)Salami500Total2008 (4.00)Total37029 (7.83)


## 3.2.P. aeruginosa Antibiotic Resistance Pattern

P. aeruginosa antibiotic resistance pattern is shown in Table 3. P. aeruginosa bacteria showed the highest resistance rate toward ampicillin (89.65%), penicillin (86.20%), and tetracycline (82.75%) antibiotic agents. The resistance rates of examined bacteria toward cefoxitin, gentamicin, clindamycin, and sulfamethoxazole were 37.93%, 34.48%, 31.03%, and 27.58%, respectively. The lowest resistance rate was observed against chloramphenicol (17.24%) and imipenem (20.68%). P. aeruginosa isolates of imported frozen bovine meat samples harbored the highest resistance against examined antibiotic agents (P<0.05). Data analysis revealed significant differences between the types of samples and the resistance rate of bacteria against antibiotic agents (P<0.05).Table 3
P. aeruginosa antibiotic resistance pattern.
Samples (N.P. aeruginosa)N. isolates resist to each antibiotic agent (%)TCSGCIPTAPCefCLNImAZFresh bovine meat (3)2 (66.66)01 (33.33)1 (33.33)1 (33.33)1 (33.33)3 (100)2 (66.66)1 (33.33)1 (33.33)01 (33.33)Frozen bovine meat (8)7 (87.50)02 (25)3 (37.50)2 (25)1 (12.50)8 (100)8 (100)2 (25)2 (25)1 (12.50)2 (25)Imported frozen bovine meat (10)10 (100)2 (20)3 (30)7 (70)5 (50)3 (30)10 (100)10 (100)5 (50)4 (40)3 (30)3 (30)Meat productsBurger (3)2 (66.66)1 (33.33)1 (33.33)2 (66.66)1 (33.33)02 (66.66)2 (66.66)1 (33.33)01 (33.33)0Kebab (4)2 (50)1 (25)1 (25)1 (25)1 (25)2 (66.66)2 (50)2 (50)1 (25)1 (25)1 (25)1 (25)Sausage (1)1 (100)1 (100)01 (100)001 (100)1 (100)1 (100)1 (100)00Total (8)5 (62.50)3 (37.50)2 (25)4 (50)2 (25)2 (12.50)5 (62.50)5 (62.50)3 (37.50)2 (12.50)2 (12.50)1 (12.50)Total (29)24 (82.75)5 (17.24)8 (27.58)15 (51.72)10 (34.48)7 (24.13)26 (89.65)25 (86.20)11 (37.93)9 (31.03)6 (20.68)7 (24.13)∗T: tetracycline (30 μg/disk); C: chloramphenicol (30 μg/disk); S: sulfamethoxazole (25 μg/disk); G: gentamicin (10 μg/disk); CIP: ciprofloxacin (5 μg/disk); T: trimethoprim (5 μg/disk); A: ampicillin (10 μg/disk); P: penicillin (10 μg/disk); Cef: cefoxitin (10 μg/disk); CLN: clindamycin (2 μg/disk); Im: imipenem (10 μg/disk); AZ: aztreonam (30 μg/disk).


## 3.3.P. aeruginosa Antibiotic Resistance Genes

Gel electrophoresis of the PCR products of virulence factors and antibiotic resistance genes is presented in Figure1. The distribution of P. aeruginosa antibiotic resistance genes is shown in Table 4. According to obtained data, blaDHA (93.10%), blaCTX-M (83.65%), and blaSHV (48.27%) were the most frequently detected antibiotic resistance genes amongst the P. aeruginosa isolates. Findings showed that blaTEM (20.68%) and gyrA (24.13%) had lower frequencies amongst the examined antibiotic resistance genes. The distribution of blaOXA and blaVEB was 34.48% and 34.48%, respectively. P. aeruginosa isolates of imported frozen bovine meat samples harbored a higher distribution of antibiotic resistance genes (P<0.05). Data analysis revealed significant differences between the types of samples and the distribution of antibiotic-resistance genes (P<0.05).Figure 1
(a) Gel electrophoresis of PCR products for the detection ofalgD and algU virulence factors. M 100 bp ladder, 1–3: positive samples for algD (275 bp) and algU (410 bp) virulence factors, PC: positive control, and NC: negative control. (b) Gel electrophoresis of PCR products for the detection of lasB, toxA, plcH, plcN, and exoS virulence factors. M 100 bp ladder, 1–9: positive samples for virulence factors and their positive controls, and NC: negative control. (c) Gel electrophoresis of PCR products for the detection of pilA, pilB, phzM, apr, phzH, phzS, exoU, and exoY virulence factors. M 1000 bp ladder, 1–8 and 10–14 and 16: positive samples for virulence factors and their positive controls, NC: negative control, and 9 and 15: negative samples for virulence factors. (d) Gel electrophoresis of PCR products for the detection of lasA, exoT, and pvdA virulence factors. M 1000 bp ladder, 1–6: positive samples for virulence factors and their positive controls, NC: negative control. (e) Gel electrophoresis of PCR products for the detection of blaTEM, blaOXA, and blaSHV antibiotic resistance genes. M 100 bp ladder, 1–6: positive samples for virulence factors and their positive controls, NC: negative control. (f) Gel electrophoresis of PCR products for the detection of the blaCTX-M antibiotic resistance gene. M 100 bp ladder, 1–3: positive samples, PC: positive control, and NC: negative control. (g) Gel electrophoresis of PCR products for the detection of blaDHA antibiotic resistance gene. M 100 bp ladder, 1–3: positive samples, PC: positive control, and NC: negative control. (h) Gel electrophoresis of PCR products for the detection of blaVEB antibiotic resistance gene. M 100 bp ladder, 1–5: positive samples, PC: positive control, and NC: negative control. (i) Gel electrophoresis of PCR products for the detection of gyrA and parC antibiotic resistance gene. M 100 bp ladder, 1–4: positive samples, PC: positive control, and NC: negative control.Table 4
P. aeruginosa antibiotic resistance genes.
Samples (N.P. aeruginosa)N. isolates harbored each antibiotic resistance gene (%)blaTEMblaSHVblaOXAblaCTX-MblaDHAblaVEBgyrAparCFresh bovine meat (3)1 (33.33)1 (33.33)1 (33.33)2 (66.66)2 (66.66)1 (33.33)1 (33.33)1 (33.33)Frozen bovine meat (8)1 (12.50)2 (25)2 (25)7 (87.50)8 (100)3 (37.50)2 (25)1 (12.50)Imported frozen bovine meat (10)3 (30)7 (70)4 (40)10 (100)10 (100)3 (30)2 (20)2 (20)Meat productsBurger (3)1 (33.33)1 (33.33)1 (33.33)2 (66.66)3 (100)1 (33.33)1 (33.33)0Kebab (4)1 (25)2 (50)2 (50)4 (100)3 (75)2 (50)1 (25)1 (25)Sausage (1)01 (100)01 (100)1 (100)000Total (8)2 (12.50)4 (50)3 (37.50)7 (87.50)7 (87.50)3 (37.50)2 (12.50)1 (12.50)Total (29)6 (20.68)14 (48.27)10 (34.48)26 (89.65)27 (93.10)10 (34.48)7 (24.13)5 (17.24)


## 3.4.P. aeruginosa Virulence Factors

The distribution ofP. aeruginosa virulence factors amongst the examined meat and meat product samples is shown in Table 5. Findings showed that exoS (75.86%), lasA (68.96%), exoU (58.62%), lasB (51.72%), plcH (48.27%), and algD (44.82%) were the most frequently detected virulence factors. Distribution of pilA, phzM, toxA, and exoT virulence factors was 24.13%, 20.68%, 17.24%, and 17.24%, respectively. PhzH (3.44%), plcN (6.89%), pvdA (6.89%), and pilB (6.89%) had the lower distribution among examined virulence factors. Statistically, significant differences were observed amid the distribution of algD and algU (P<0.05), plcH and plcN (P<0.05), exoS and exoT (P<0.05), exoU and exoT (P<0.05), exoS and exoY (P<0.05), exoU and exoY (P<0.05), phzM and phzH (P<0.05), lasA and lasB (P<0.05), and pilA and pilB (P<0.05). P. aeruginosa isolates of imported frozen bovine meat samples harbored a higher distribution of virulence factors (P<0.05). Data analysis revealed significant differences between the types of samples and the distribution of virulence factors (P<0.05).Table 5
P. aeruginosa virulence factors.
Samples (N.P. aeruginosa)N. isolates resist each virulence factor (%)algDalgUtoxAplcHplcNexoSexoTexoYexoUaprphzMphzSphzHlasAlasBpvdApilApilBFresh bovine meat (3)1 (33.33)01 (33.33)2 (66.66)02 (66.66)1 (33.33)02 (66.66)00002 (66.66)2 (66.66)01 (33.33)0Frozen bovine meat (8)3 (37.50)1 (12.50)1 (12.50)3 (37.50)06 (75)1 (12.50)04 (50)1 (12.50)1 (12.50)1 (12.50)05 (62.50)3 (37.50)01 (12.50)0Imported frozen bovine meat (10)5 (50)2 (20)2 (20)6 (60)2 (20)8 (80)2 (20)2 (20)7 (70)2 (20)3 (30)1 (10)1 (10)8 (80)6 (60)1 (12.50)4 (40)1 (12.50)Meat productsBurger (3)1 (33.33)001 (33.33)02 (66.66)1 (33.33)01 (33.33)01 (33.33)002 (66.66)1 (33.33)001 (33.33)Kebab (4)2 (50)1 (25)1 (25)1 (25)03 (75)01 (25)2 (50)1 (25)1 (25)1 (25)02 (50)2 (50)1 (25)1 (25)0Sausage (1)1 (100)001 (100)01 (100)001 (100)00001 (100)1 (100)000Total (8)4 (50)1 (12.50)1 (12.50)3 (37.50)05 (62.50)1 (12.50)1 (12.50)4 (50)1 (12.50)2 (25)1 (12.50)05 (62.50)4 (50)1 (12.50)1 (12.50)1 (12.50)Total (29)13 (44.82)4 (13.79)5 (17.24)14 (48.27)2 (6.89)22 (75.86)5 (17.24)3 (10.34)17 (58.62)4 (13.79)6 (20.68)3 (10.34)1 (3.44)20 (68.96)15 (51.72)2 (6.89)7 (24.13)2 (6.89)


## 4. Discussion

An emerging food bacterium,P. aeruginosa is a frequent opportunistic human pathogen that is extensively found in the environment; it plays an essential portion in the spoilage of different foods [30]. Thus, it is essential to determine its prevalence and other epidemiological properties.In the present study, 7.83% of meat and meat product samples were contaminated withP. aeruginosa. A higher prevalence rate of P. aeruginosa was reported in imported frozen bovine meat (20.00%) and frozen bovine meat (13.33%). Considering the psychrophilic nature of the bacterium, it is logical that the frozen samples had higher bacterial contamination. The high contamination rate of imported meat samples may be due to the importation of low-quality meat or their long storage period in customs in harmful conditions. Improper application of heat chain and cooking time and contamination in samples of meat products after production can be possible reasons for P. aeruginosa in these samples.Sheir et al. [31] described that P. aeruginosa was detected in 4.00% of frozen burger samples. They revealed that unnecessary handling, malpractices, poor hygienic quality of raw materials, particularly spices, and unsanitary practices during production and storage were the main reasons for P. aeruginosa in frozen meat samples. Benie et al. [32] reported that the P. aeruginosa prevalence amongst smoked fish, fresh fish, and bovine meat samples collected from West Africa was 23.57%, 37.69% 53.04%, respectively. Up to now, the highest P. aeruginosa prevalence among food samples has been reported by Benie et al. [33]. They disclosed that the Pseudomonas species prevalence was higher in beef (97.90%), followed by fresh fish (87.80%), and smoked fish (63.10%). P. aeruginosa prevalence amongst the chicken meat [34], camel meat [35], retail meat [36], frozen imported meat [37], and sausage [38] samples was 46.70%, 80.00%, 3.00%, 6.67%, and 8.33%, respectively. Highly distribution of P. aeruginosa in examined samples may be due to high P. aeruginosa adaptation to a different environment, ambient temperatures (4 to 42°C), and low water activities (72%–97%) [39]. Thus, it is crucial to consider this bacterium in meat and meat products.In addition to the high prevalence of bacteria in the studied meat and meat product samples,P. aeruginosa was also highly resistant to most of the evaluated antibiotics, especially ampicillin, penicillin, tetracycline, cefoxitin, gentamicin, clindamycin, and sulfamethoxazole. The high prevalence of antibiotic resistance amongst examined bacteria was also assisted by the high distribution of antibiotic resistance genes, especially blaDHA, blaCTX-M, and blaSHV. Similarly, a high prevalence of resistance toward such antibiotic agents was reported by Meng et al. (China) [40], Khan et al. (Australia) [41], Yayan et al. (Germany) [42], and Khan and Faiz (Saudi Arabia) [43]. In a survey conducted by Benie et al. [32], P. aeruginosa isolates of meat samples revealed the highest resistance rate toward kanamycin (100%), aztreonam (97.60%), and ciprofloxacin (35.40%). Allydice-Francis and Brown [44] stated that the P. aeruginosa isolates of vegetable samples harbored a high resistance rate toward ceftazidime (79.00%), ciprofloxacin (93.00%), gentamicin (97.00%), and imipenem (100%) antibiotic agents. Odumosu et al. [45] mentioned that the P. aeruginosa isolates of ready-to-eat vegetable samples collected from Nigeria were resistant against carbenicillin (63.00%), amikacin (1.90%), and ceftazidime (83.30%). P. aeruginosa strains isolated from imported frozen meat samples harbored a higher distribution of antibiotic resistance. One possible explanation for this finding is the importation of low-quality meat samples from other countries to Iran. Additionally, the addition of antibiotic resistant P. aeruginosa strains from other foodstuffs into imported meat samples and during storage are other probable reasons. Chloramphenicol is a prohibited antibiotic agent. Resistance against this antibiotic may show its unauthorized prescription in veterinary. In this survey, resistance toward chloramphenicol was obtained in 20% of P. aeruginosa strains isolated from imported frozen bovine meat, 33.33% of burger-based isolates, 25% of kebab-based isolates, and finally all (100%) of strains isolated from sausage samples. This finding may indirectly show the application of poultry products in the production of sausage, burger, and kebab samples.Irregular and irrational administration of antibiotics in veterinary medicine is the probable reason for antibiotic resistance in bacteria. Additionally, it could be transferred by plasmids and transposons. In the food systems, ESBL-producingP. aeruginosa occurrence is emerging [46]. Our findings revealed that the genes that encode resistance toward all beta-lactams (blaShV, blaDHA, blaVEB, blaCTX, blaTEM, and blaOXA), and quinolones (gyrA and parC) were predominant amongst isolated bacteria. In keeping with this, as we did not perform the ESBL production test, we cannot exactly mention whether the isolated strains are ESBL producers or not. In another Iranian survey, Bahrami et al. [47] described that blaCTX-M, blaSHV, blaTEM, and blaOXA were the most frequent antibiotic resistance genes detected in 23.95%, 23.08%, 57.29%, and 12.5% of P. aeruginosa strains, respectively. A survey on camel meat [48] showed that the total distribution of ESBL-producing P. aeruginosa was 45%, and blaCTX-M (38.00%), blaSHV (33.30%), and blaTEM (28.50%) were the most commonly detected antibiotic resistance genes. Extended-spectrum b-lactamases are encoded by bla plasmid-mediated genes, particularly blaCTX-M, blaSHV, blaTEM, blaVEB, and blaOXA, which have extended the substratum specificity toward cefotaxime, ceftazidime, and ceftriaxone [49]. Hosu et al. [50] stated that blaSHV, blaTEM, and blaCTX-M antibiotic resistance genes amongst the P. aeruginosa isolates of nonclinical samples were distributed 93.30%, 40.00%, and 20.00%, respectively.Another critical aspect of theP. aeruginosa presence in examined samples was the high distribution of virulence factors, particularly exoS, lasA, exoU, lasB, plcH, algD, pilA, phzM, toxA, and exoT. These genes are mainly responsible for the adhesion and invasion of bacteria into the host cells. In keeping with this, consuming food containing virulent P. aeruginosa strains may cause severe food-borne infection. ToxA, aprA, plcH, and las virulence factors were predominant in the P. aeruginosa strains isolated from camel meat [35]. Benie et al. [32] recognized that the distribution of exoU, pilB, plcH, algD, exoS, and lasB virulence factors in the P. aeruginosa strains isolated from bovine meat samples were 4.10%, 42.60%, 72.10%, 74.50%, 96.70%, and 96.70%, respectively.The highlasA and B genes distribution specifies that these protease enzymes can be significant for the P. aeruginosa pathogenesis cutting collagen and elastin [51]. It also can destruct the junctions amid the epithelial cells [52]. LasA and lasB genes increase IL-8 production and decrease the innate immune response, immunoglobulins, and complement compounds [51, 52]. The high algD distribution caused alginate overproduction. Its high distribution maybe shows that isolated strains of meat and meat product samples are involved in biofilm formation as alginates have been extensively regarded as the main biofilm matrix exopolysaccharides [53]. It can protect the bacteria from antibiotics, decrease phagocytosis and polymorphonuclear chemotaxis, and inhibit complement activation [53]. ExoS and plcH virulence genes were also detected in 75.86% and 48.25% of P. aeruginosa isolates. Thus, the isolates can secrete hemolytic exoenzymes and phospholipase C, which can facilitate the occurrence of some kinds of human clinical infections [54]. The exoS was responsible for bacterial dissemination and tissue destruction [54]. The pilA, pilB, and exoU genes were detected in 24.13%, 6.89%, and 58.62% of P. aeruginosa isolates. Both pil genes are responsible for pili formation and further motilities [55]. The ExoU gene is cytotoxic, and its distribution is a marker of invasive P. aeruginosa [56]. It seems that P. aeruginosa isolates of meat and meat product samples in this survey can cause severe gastrointestinal disorders owing to the high distribution of virulence factors. However, some additional works should be performed to approve it.


## 5. Conclusion

In assumption, the highP. aeruginosa prevalence in samples predominantly imported frozen bovine meat and frozen bovine meat samples with a high distribution of virulence factors and antibiotic resistance was reported in this study. A high prevalence of resistance to ampicillin, penicillin, tetracycline cefoxitin, sulfamethoxazole gentamicin, and clindamycin antibiotic agents and the presence of BlaDHA, blaCTX-M, blaSHV, BlaTEM, and gyrA antibiotic resistance genes were reported in the present survey. These findings may show the high antibiotic resistance of P. aeruginosa and the potential role of meat and meat products in its transmission to the human population. Some strains harbored different virulence factors, particularly exoS, lasA, exoU, lasB, plcH, algD, pilA, phzM, toxA, and exoT. These genes are mainly involved in the adhesion and invasion to host cells. It seems that the consumption of contaminated meat and meat products with virulent P. aeruginosa may cause severe food-borne diseases. Additional investigations are suggested to illuminate additional epidemiological aspects of antibiotic resistant and virulent P. aeruginosa in raw meat, frozen meat, imported frozen meat, and meat product samples.



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