Introduction

Many people in Nineveh province consumed local cheeses using raw milk. During this process, cheese was exposed to contamination from the milk, handler, and surrounding environment during preservation under chilling temperature 4°C. P. fluorescens as a psychotropic bacterium, may grow and multiply in many foods such as milk and cheese and become the dominant microflora displaying spoilage defects, including changes in color, odor-flavor, and texture, which reduce the shelf life of cheese and affect the quality (1,2). These bacteria have been reported in many types of cheeses (3) P. fluorescens was the most specific spoilage microorganisms of milk and dairy products during storage under refrigeration temperatures (4) it is highly spread and can enter the dairy products plants through post-pasteurization contamination, it is already present in soil, dust, and water in small fraction within dairy animal environments (5). The high genomic diversity of P. fluorescens strains needs a genetic approach to confirm the strains, such as 16srRNA sequencing (6-8). Many strains of P. fluorescens produce extracellular heat-stable protease, lipase, and lecithinase, which contribute to the spoilage of milk and milk products, including cheese (9). Their activities degrade milk constituents such as casein, when protease digest casein, milk gelatin will occur and milk fat will be hydrolyzed (10,11). An alkaline zinc metalloprotease has a molecular mass of about 42 Kilo Dalton (12,13). Also, P. fluorescens can produce pigments such as pyoverdin, fluorescein, and pyomelanin, which cause food discoloration (14). Several studies highlighted the effect of thermo-resistant protease on the sensorial features of cheese (15,16). Some studies screen the bacterial contamination of milk, and cheese and the antimicrobial susceptibility of pseudomonas (17-19).

There was no study on the protease and lipase activity of P. fluorescens from milk and cheese in Nineveh province, therefore, the current study was designed to monitor the existence of P. fluorescens in local cheese and their abilities to cause cheese spoilage according to protease and lipase activity in using conventional polymerase chain reactions assay.

Material and methods

Ethical approval

All samples were obtained after the owner’s approval, and the research was carried out according to the ethical guidelines of the Institutional Animal Care and Use Committee at the College of Veterinary Medicine, University of Mosul, which included an authorized ID of UM.VET. 2023.083.

Samples

The study included 50 samples of local fresh cheese collected randomly from different regions in Nineveh province between Oct. 2023 and Mar. 2024.All samples were preserved in an ice box and then transported to the Laboratory of Veterinary Public Health, College of Veterinary Medicine, University of Mosul.

Isolation

Cheese samples were examined to isolate psychotropic P. fluorescens on Pseudomonas cetrimide agar (Neogen, USA). Plates were incubated at 25°C for two days, and colonies were identified according to phenotypic characteristics (20).

Identifications of bacterial isolates

The identification of P. fluorescens isolates was related to some biochemical tests, including the Oxidase test, Catalase test, Starch hydrolysis, pyoverdine production, and Gelatin liquefaction abilities (21). Molecular identification of P. fluorescens was used to confirm the diagnosis using a polymerase chain reaction assay (PCR).

DNA Extraction

According to the manufacturer’s profile, suspected colonies were subjected to DNA extraction using a Bacterial DNA kit (Add a bio, Korea).

Polymerase chain reaction (PCR)

The P. fluorescens strains isolated from cheeses were confirmed depending on PCR assay using the 16SPflu gene, a universal primer provided by (Macrogen/Korea). The primer consists of forward and reverse primer sets following with a molecular weight of 850 bp. The products exposed to a thermal profile included denaturation of 2 min. at 95ºC then 35 cycles of 94ºC for 45s, followed by annealing 56ºC for 60 s. and extension at 72ºC for 1 min. and final extension at 72ºC for 2 min. with cooling at 4ºC. The products were illustrated by electrophoresis (1.5% agarose gel) manufactured by (AddBio, Korea) with three μl GelRed dye (AddBio, Korea). The PCR products were analyzed in 300mA 75 volts for 1 hour. 5 μl of DNA ladder with 100 base pairs (GeNet Direx, Korea) was standard. The specific band of DNA was identified using the gel documentation system (Bio-Rad, USA). The positively identified strains of P. fluorescens were screened to detect their protease and lipase activity depending on AprX and LipM genes with product size of 1434 and 1422 base pairs, respectively (Table 1), the PCR reactions done according to manufacturer instructions.

Sequencing of the 16SrRNA gene

After the PCR products were purified, the sequencing of the 16SPflu gene was assessed according to Sanger dideoxy sequencing and the Blast algorithm at the NCBI server. Then phylogenic analysis was done using ClustalX (NCBI) software programs [available at]. The phylogenetic tree structure was done using the Maximum Likelihood approach depending on the Tamura-Nei model in MEGA11 software.

Table 1: Oligonucleotide Primers sequence for P. flouresence used in the current study

Results

The results revealed a successful recovery of P. fluorescens strains in local fresh cheese (14/50) %28 by conventional culture methods and (10/50) %20 was positive for P. fluorescens strains using PCR techniques (Table 2). The PCR results confirmed the detection of P. fluorescens isolates according to the 16SrRNA gene producing bands with 850 base pairs (Figure 1). Additionally, screening of P. fluorescens protease activity was detected only (3/10) strains at (%30) according to the presence of AprX gene producing bands at 1434 bp and (1/10) 10% for the existence of LipM gene with amplicon 1422 bp (Figure 2 and 3). Sequencing of the 16SPflu gene exhibits that strains of P. fluorescens isolated from cheese have been submitted to the Genebank database with accession numbers PP727372, PP727373, PP727374, and PP727375 were registered in the National Center for Gene Bank. The alignment of local P. fluorescens with NCBI GenBank is shown in (Figure 4). According to Blast, the local isolates accession number matches the China isolates of P. fluorescens OP341878, MW582677 gene with a percentage of 100% (Table 3). The relationship between Iraqi local isolates and global isolates was obtained according to the phylogenic tree using the Maximum Likelihood approach depending on the Tamura-Nei model in MEGA11 software. The P. azotoformans (MT998034-Austria) were rooted as outgroup (Figure 5).

Table 2: The prevalence of P. fluorescens in local fresh cheese by conventional and PCR assay

Figure 1: Genomic characterization of PCR products for the 16SPflu gene of Pseudomonas fluorescens, M lane represents a 100 base pair DNA ladder. Lanes 1-10 are positive cheese samples at 850 base pairs, and lane 11 is a negative control.

Figure 2: Genomic characterization of PCR products for the AprX gene of Pseudomonas fluorescens. M lane represents a 100 base pair DNA ladder. Lanes 1,8, and 9 are positive samples at 1434 base pairs, lanes 2-7 and 10 are negative samples, and lane 11 is a negative control.

Figure 3: Genomic characterization of PCR products for the Lip gene of Pseudomonas fluorescens. M lane represents a 100 base pair DNA ladder. Lane 10 has positive samples at 1422 base pairs, lanes 1-9 and 11 are negative samples, andlane 12 inegative control.

Figure 4: Identifying the query sample, Pseudomonas fluorescens, by alignment in NCBI gene bank.

Table 3: Percentage distribution of P.  fluorescens based on 16SrRNA gene according to BLAST in GenBank of NCBI

Figure 5: The phylogenic tree of P. fluorescens from cheese in Nineveh province is pointed in a red circle. The P.azotoformans (MT998034-Austria) represent the outgroup.

Discussion

The source of the milk used for cheese manufacture is essential factor affecting final products. psychotropics bacteria are involved in the defects in dairy products due to prolonged chilling storage (24,25). Pseudomonas spp. is one of the most common microbiotas in raw milk under cold storage. Physicochemical traits of fresh cheeses are suitable for the growth of P. fluorescens, especially the a^w and pH (26,27). Our results confirmed the findings of other studies on the prevalence of P. fluorescens in cheese in Australia (28,29) and from Damietta cheese in Egypt at 35.14% (30). which may attribute to the Hydrolysis of casein liberate plasmin and plasminogen which altering the cheese yield and affecting the sensory traits of the final product of cheeses (2,31,32) or may be attributed to the microbiota of milk supplied for cheese production and affect cheese quality. The composition of cheese and pH may affect proteolysis patterns with cheese hardness, which affects cheese texture and flavor (33-35). Similarly, a thermoresistant protease produced by a P. fluorescens strain hydrolyzed β-casein in milk increasing protease activity over storage time before cheese processing (16,36,37). Higher ripening pH and temperature affect the protease activity of P. fluorescens in cheese Both AprX and LipM genes are depended on as an indicator to detect the virulence of P. fluorescens to induce spoilage (38,39) and by using PCR assay as a more flexible method for early detection of P. fluorescens to predict the shelf life of the products (40).The study revealed the prevalence of P. fluorescens in dairy chains in Nineveh province as mentioned in the Genebank database using partial specific region genetic sequencing of 16srRNA of P.fluorescens isolated from local cheese for the first time in my city, the comparative. study of our local strains with global strains recorded in Genebank database referred to highly aggregation cluster of local isolates indicating transmission due to contamination as well as the essential role of environmental effects on this genetic diversity where the flexibility of the Pseudomonas genome, permitting the accession of nutrient-scavenging pathways through variant environments (41). The 99% identity may be attributed to mini nucleotide differences from world strains due to mutation (42). Therefore, adequate hygienic conditions should be provided to reduce bacterium growth and reduce dairy product spoilage.

Conclusion

The detection of P. fluorescens in local fresh cheese indicates the possibility of spoilage by microorganisms arising from contaminated milk supplies for manufacturing. The risk of these bacteria comes from the liberation of protease and lipase which accelerate spoilage and reduce cheese shelf life. Therefore, due to their high diversity we need to restrict the growth of pseudomonas spp. in dairy products to minimize spoilage and maintain cheese quality.

Acknowledgments

This research was supported using resources from the University of Mosul's College of Veterinary Medicine in Mosul, Iraq.

Conflict of interest

The authors confirm there was no conflict of interest.