Antibiotics resistance patterns of Pseudomonas aeruginosa isolated from meat at Mosul city retails

1. Molecular detection of pseudomonas aeruginosa from meat using rpoB gene.
2. Monitoring the antibiotics resistance patterns of pseudomonas aeruginosa isolated from meat at Mosul city retails.
3. The challenge of pseudomonas aeruginosa from meat to antimicrobials. 

Introduction

Meat quality is a multifaceted topic influenced by internal and external animal variables. The consumer recognizes quality through good sensory attributes. To avoid economic losses, the producer must meet organoleptic conditions; and ensure that the product remains microbiologically safe during storage (1). Meat spoiling is caused by a range of factors, including oxidation, enzymatic autolysis, and microbiological growth, which vary based on husbandry practices, animal species, ages at slaughtering, handling, processing, and preservation method. Microbial spoilage leads to pH alteration, degradation, and slime formation, reflecting off odor and loss of bloom (2,3). Pseudomonas is a significant gram-negative bacterium that spread widely in food, water, soil, and the environment due to their simple nutritional requirements. In addition, it has been related to human and animal illness and a high mortality rate in broiler chickens (4), it is an excellent concern in newly hatched chicks (5). It also has a high distribution within the food ecosystem (6). Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas fragi are the most representativespecies of the genus in animal products, Pseudomonas has a significant value in meat and milk because of its ability to produce several lipolytic and proteolytic enzymes and develop a biofilm that impacts food quality (7-9). P. aeruginosa is an opportunistic pathogen associated with meat spoilage and reduces the shelf life of meat and meat products (10-12). It is associated with severe infections, especially in immunocompromised individuals (13,14). A single flagellum and multiple cell surface pili offer bacterial movement and adhesion characteristics, allowing pseudomonas species to colonize surfaces, especially under cold and aerobic conditions (15,16). P. aeruginosa causes many infections that do not respond to treatment and are life threatening due to their high multi-drug resistance to antibiotics through their lower outer membrane permeability (17,18). The production of antibiotic-inactivating enzymes and the expression of efflux-pumps that expel the antibiotics out of the cell propagate antimicrobial resistance genes across the food chain, all of them increasing the resistance gene pool and posing a risk to the public health (19,20). Clinical isolates of P. aeruginosa possess a multidrug-resistance than the food and environmental isolates. Antimicrobial resistance of a prospective disease to a wide range of antibiotics could signal a severe problem that affects public health (21,22). Because there has been no previous research on the antimicrobial activity of P. aeruginosa isolated from meat, the current study was designed to evaluate the prevalence of P. aeruginosa in meat displayed in Mosul city retails and assess its antibiotic resistance patterns.

Material and methods

Ethical approval

The scientific committee of the veterinary public health department approved this work on the twelfth session at 20/June/2021.

Samples

The study was based in Mosul city, and meat samples were taken from different markets and butcher’s shops. One hundred fifty meat samples from beef, mutton, and chicken each 50 were collected randomly and under sterile conditions the samples were transported as soon as possible to the College of Veterinary medicine, Veterinary Public Health Laboratory, for further microbiological analysis.

Isolation and identifications

Meat samples were homogenized with nutrient broth under sterile conditions for enrichment. Additionally, cetrimide agar medium (Neogen, USA) was used to isolate Pseudomonas as a selective medium, Plates were inoculated using the streaked plate method and incubated aerobically at 37°C for 24 hours, and the pure culture of P. aeruginosa was exposed to ultra-violet light to detect the fluorescence ability. Detection of P. aeruginosa isolates was done microscopically based on using a light microscope to observe Gram staining, confirmed by biochemical tests (23,24). Molecular detection of P. aeruginosawere done usingthe rpoB gene to confirm the isolates (25).

Antibiotic susceptibility test

To assess the susceptibility ofP. aeruginosaisolates to antibiotics, the standard Kirby-Bauer disk diffusion method was used according to Jorgensen and Turnidge (26). The antibiotics discs (Bioanalyse, Turkey) used against pseudomonas isolates including levofloxacin (LEV 5 μg), ciprofloxacin (CIP 5 μg), amoxicillin/clavulanic acid (AMC 30 μg), piperacillin (PRL 100 μg), aztreonam (ATM 30 μg), meropenem (MEM 10 μg), tobramycin (TOB 10 μg), gentamicin (CN 10 μg). The suspension of isolates equivalent to 0.5 McFarland opacity standard was prepared and inoculated on Mueller-Hinton agar plates using a sterile swab. Then, the discs were placed aseptically on the inoculated surface of the media and incubated for 24 hours at 37ºC. After the incubation period, the diameter of the inhibition zone to each disc was measured using a digital caliper (Ingco, China). The results were translated to Susceptible, Intermediate, and Resistant categories by comparing inhibition zone diameters according to manufacturer instructions.

Statistical analysis

Statistical analysis of the observed data was done with a Chi-square test using SPSS software version 22.

Results

Depending on molecular detection of P. aeruginosa according to the presence of the rpoB gene by conventional PCR, out of 150 meat samples collected from Mosul city retails, twenty-one isolates of P. aeruginosawere positive with a total recovery rate pf 14% (Table 1). The results revealed significant differences between the recovery rate of P. aeruginosafrom beef and chicken meat compared to mutton at P

Table 1: The prevalence of Pseudomonas aeruginosa in different types of meat

Figure 1: (a) growth of P. aeruginosa on cetrimide agar, (b) fluorescence activity under UV-light.

Among the 21 isolates of P. aeruginosa from different types of meat, the antimicrobial resistance profile revealed high resistance of isolates to amoxicillin at 100%, aztreonam at 66%, while meropenem was recorded at 14%. In contrast, P. aeruginosa isolates were susceptible to tobramycin 100%, levofloxacin 90%, ciprofloxacin 90%, gentamicin 90%, followed by piperacillin 76% and meropenem 57%. at various levels, some strains showed a multi-drug resistance to amoxicillin, aztreonam, and meropenem (Figure 2).

Figure 2: Antibiotics resistance patterns of P. aeruginosa isolated from meat

Discussion

Pseudomonas is a potent indicator of food spoilage, especially in meat and meat products leading to putrefaction under aerobic conditions (27). Meat processing can be the primary source of contamination in fresh meat and meat products that are not exposed to heat during preparation (28). Our results exhibited 14% P. aeruginosa recovery rate in meat displayed at Mosul city retails, which may be related to defects in storage temperature. These results agree with the recovery rate of P. aeruginosa reported by Hemmat (29) in frozen imported meat. In contrast the prevalence of P. aeruginosa in bovine meat was 47. 8% in Abidjan (30). The differences in the prevalence of P. aeruginosa in beef, mutton, and chicken meat may be related to the initial contamination level. Intrinsic properties of meat despite the variation in the recovery rate of P. aeruginosa from animal sources, the presence of these microbiotas in meat reflects the weakness of hygienic measurement applied in meat production and marketing, which negatively impacts meat quality and accelerates spoilage (31,32). The low isolation rate of P. aeruginosa in mutton may be due to the elevation of other microbiota such as p. fluorescens andenterobacteriaceaethrough competition. Also, a higher intramuscular fat content of mutton reduces the growth of spoilage bacteria, especially if it was preserved under vacuum packaging (33,34). The antibiotics resistance patterns of Pseudomonas varied according to phylogeny. The P. aeruginosastrains isolated from different types of meat were sensitive to aminoglycosides, fluoroquinolones, penicillin and carbapenems with total isolates resistance to amoxicillin and high resistance to aztreonam. These results agree with (35,36) who conducted that the resistance to beta-lactams was related to the presence of plasmid-mediated extended-spectrum beta-lactamases. Another study conducted by Murphy et al. (37) on frozen meat accepts our findings. Moreover, 29% of isolates show intermediate resistance to meropenem, this high percentage of resistance may be related to the indiscriminate use of antibiotics by veterinarians in farm animals as treatment or as growth promoters and can be transmitted to human beings (38,39). The most effective antibiotics against P. aeruginosa weretobramycin, levofloxacin, ciprofloxacin and gentamicin similar to pseudomonas resistance recorded in poultry meat by Kousar et al. (40). Also, poultry farms spread multi-drug resistance in their environment (41,42). Both Piperacillin and Meropenem are the second most effective antibiotics against P. aeruginosa from meat. Our results were approved by Glen and Lamont (43) who reported ciprofloxacin as a potent drug against Pseudomonas. Due to the excessive use of antibiotics in livestock farms, a severe public health hazard is developed. Therefore, possible strategies are essential for enhancing the effectiveness of antibiotics, especially the beta-lactam. in treating p. aeruginosa infections (44,45). Finally, proper handling of raw products, adequate cooking of meat, and proper disposal of waste, can minimize the antibiotics resistance of these bacteria, which may reflect the misuse of antibiotics in animals.

Conclusion

Antimicrobial resistance is a significant challenge to food safety and consumer health. The antibiotic resistance of P. aeruginosa may generate a burden and limit the choice of antibiotics due to resistance development.

Acknowledgments

The University of Mosul in Mosul, Iraq. The College of Veterinary Medicine supported this research.

Conflict of interest

The authors declare that there is no conflict of interest.