Molecular Characterization and Biofilm Formation Study of Contaminant Bacteria Isolated from Domiaty and Hungarian Cheeses in Jeddah City

The aim was to study the microbiological quality of Domiaty and Hungarian cheeses, molecular identification and biofilm formation of some selected contaminant bacteria. Samples were collected from two M and P big markets in Jeddah City through the period from February to October 2018, nine visits for two types of natural cheese. Results showed that the total bacterial counts (CFU/ml) from Domiaty cheese from two markets (M and P) were 0.1 x 105, 8 x 105 and 1 x 10 5 CFU/ml respectively (3 visits of M market) and 4 x 106, 0.4 x 106, 6.5 x 103, 1 x 103, 0.1 x 103 and 0.1 x 103 CFU/ml respectively (six samples from 6 visits from P market). Results showed that the total bacterial counts (CFU/ml) from Hungarian cheese were 1.5 x 10 5, 1x 10 4, 11 x 10 4 and 4 x10 6 CFU/ml respectively from (4 visits of M market) and 0.18 x 104, 3 x 106, 22 x 106, 6 x 106 and 5 x 104 CFU/ml respectively (5 visits from P market).Different bacterial isolates from cheese were identified by morphology and biochemical test. Bacterial isolates from cheeses were identified by VITEK MS as follow: Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2), and Pseudomonas fluorescens (D14-2) were isolated from Domiaty cheese while Enterococcus faecium (H11-2), Serratia liquefaciens (H15-1) and Streptococcus thermophilus (H14-1) were isolated from Hungarian cheese. Some selected bacterial isolates were identified by 16S rRNA. Isolates were belong to MK757978 (Raoultilla terrigena (D15-1)), MK757979 (Bacillus cereus (D16-1)), MK757980 (Enterococcus faecalis (H10-2)), MK757982 (Enterococcus fiscalism (H11-1)), MK757981 (Serratia liquefactions (H13-1)), MK757984 (Anoxybacillus flavithermus (H17-1). All bacterial isolates have been tested for the formation of biofilm using a Tissue Culture Plate (TCP). Results revealed 12.5% and 46.15% of high biofilm formation respectively for bacterial isolates of Domiaty and Hungarian cheeses.


INTRODUCTION
The food is the fuel of our life and it is a major concern for quality and safety 1 . Cheese is most common in Saudi Arabia, because of its health benefits and flavor, also it is a rich source of dietary calcium, proteins, and phosphorus 2 .
The microbial contamination in the cheese may arise from different sources, these sources during the cheese production as: ground, starter culture, brine, packaging materials, cheese cloth, yogurt cut knife, cold room and air room production (Temelli et al).
There are several factors responsible for Domiaty cheese microbiological quality such as the thermal treatment of the milk, the raw milk, and the level and type(s) of microbial contamination that occur throughout the manufacture and cheese storage as reported by Bintsis and Papademas 3 . Domiaty cheese is one of the most popular varieties of cheese, if contaminated, it causes of foodborne illness. Cokal et al 4 reported that (Staphylococcus aureus, Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes) were foodborne pathogens that the most common and responsible to outbreaks associated with cheese.
According to 5 , the cheese should be free from pathogens such as, Staphylococcus aureus, Salmonella sp, Clostridium botulinum, Listeria monocytogenes, Campylobacter jejuni, Bacillus cereus, Escherichia coli O157:H7, Sreptococcus faecalis and indicator hygiene include Coliform group and fungi shouldn't exceed 10 cfu/g and the yeast shroud not exceed 400 cfu/g. according to the manufacturing processes, there are many subtypes of Domiaty cheese.
Different factors, control growth pathogens on cheese include organic factor, PH value, moisture, salt concentration, temperature and hygienic control on the diary plant 6,7 . Cheese consider as a good bacterial growth medium due to the content of nutrients and long storage duration, and several steps in production may cause bacterial risks 8 . Cheese contamination can occur with foodborne pathogens in several stages in cheese processing, as pastoralized milk, row milk, after pastoralized milk 9 . Foodborne pathogens contaminated different types of cheeses as Staphylococcus aureus, Listeria monocytogenes, Salmonella spp. and E. coli. S. aureus, Salmonella spp. or E. coli can be transferred by Food-borne outbreaks occur from eating food contaminated with these pathogens that lead to serious illness 10 . Several lactic acid bacterial species from Domiaty cheese were isolated and identified, such as Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcus lactis subsp. lactis, L. casei as reported by Fahmy and Youssef 11 and Enterococcus faecalis, E. faecium and L. farciminis, L. alimentarius as reported by El-Zayat et al 12 and EL-Hamshary et al 13 isolated different bacterial strains from white cheese B. cereus (S1) Staphylococcus aureus (S2); Bacillus paramycoides (S3); Staphylococcus aureus (S5); Serratia proteamaculan (S6); Serratia proteamaculan (S7) and Serratia proteamaculan (S9)).
A biofilm consists of one or more of bacterial strains in extracellular polymeric substance (DNA, protein or carbohydrates) matrix 14 , or as reported by Satpathy et al. that bacterial strains bind to surfaces and form spatially structured communities inside a self-produced matrix, containing extracellular polymeric substances (EPS) known as biofilms. Also, Wingender and Flemming 15 reported that extracellular polymeric substances (EPS) are biosynthetic polymers produced by microorganisms from prokaryotic, and the production of EPS by bacterial strains in (culture or aggregates) is affected by the microbial species, phases of growth, nutritional status and the conditions of environment 16 . Bacterial EPS affect cell adhesion, microbial aggregates formation (biofilms, flocs, sludges and bio-granules), as reported by Comte et al 17 ). Biofilms are very important for the industry of food because biofilms make bacteria to bind to a number of surfaces, including food products, rubber, polypropylene, plastic, glass, stainless steel, and through just a few minutes, then is followed by mature biofilms developing (a few days or hours) 18 , Food processing lines are a suitable environment for biofilms to form on food contact surfaces, primarily due to manufacturing plants' complexity, long production periods, mass product generation, and large biofilm growth areas 19 . Many food-borne bacteria may, therefore, bind to the contact surfaces present in these areas, which could contribute to increase the risk of bacterial food-borne diseases. 80% of bacterial infections for example in the USA are believed to Journal of Pure and Applied Microbiology be related specifically to food-borne pathogens in biofilms 20 .
In the industry of food, species that forming biofilm appear in environments of factory and can be pathogenic to humans because they develop biofilm structures. The processing environments of the food industry, e.g., wood, glass, stainless steel, polyethylene, rubber, polypropylene, etc., act as artificial substrates for these pathogens as reported by Abdallah et al 21 and Colagiorgi 22 .
The characteristics of attachment surface's affect the production of mixedspecies biofilm 23 , conditions of environment 24 , and involved bacterial cells 25,26 . Food matrix components 27 , in food processing environments also influence attachment of bacteria 28 ; e.g., food waste, such as exudates of milk and meat enriched in fats, carbohydrates and proteins, facilitate microorganism multiplication and growth, and favors dual-species biofilm development by E. coli and Staphylococcus aureus 29,30 reported that milk lactose improves biofilm production by Bacillus subtilis, by activating the LuxS-mediated quorum-sensing system, and S. aureus through development intercellular polysaccharide adhesion 31 .
Lafarge et al 32 detected Serratia spp. bacterial strains in different sources as raw milk, in a milk-processing plant 33 , milk bulk tank as reported by Decimo 34 , and from internal surfaces of tankers of raw milk and reported that produce (heatresistant proteolytic enzymes) and it is included in monitoring the refrigerated raw milk quality, and biofilms producer in single culture and in mixed with Streptococcus uberis on the stainless-steel surfaces 35,36 , and Serratia spp. possess forming biofilm much higher than for Pseudomonas spp. and showed that Serratia isolates were found as one of the most predominant proteolytic enzymes producers Pseudomonas spp. biofilms tended to have a smaller ratio of mass: cells and mixed with Serratia spp., presenting the opposite pattern as reported by Cleto et al 33 . The presence of a single different strain may have a significant effect on the microbial dynamics in dairy products 32 .
Machado et al 37 reported that in dairy products, the dynamics of a microbial population have been studied by molecular methods, based on sequencing a fragment of 16S rDNA gene and comparing with NCBI databases. The most proteolytic isolates were selected for identification using 16S rDNA sequencing. Serratia liquefaciens (73.9%) and Pseudomonas spp. (26.1%) were identified as the dominant psychrotrophic microorganisms with high spoilage potential. The milk spoilage microbiota knowledge will be important for improve milk and dairy products quality. Serratia liquefaciens is a spoilage microorganism of relevance in the dairy industry because it is psychrotrophic, biofilm producer, and produces thermoresistant lipases and proteases 38 , and from milk as showed by Gaffer et al 39 .
Bacillus cereus is a Gram-positive and spore-forming bacterium that can grow in various environments at wide-ranging temperatures (4°C-50°C), and It is resistant to (chemicals, radiation and heat treatment) 40 . Pathogenic bacteria as Bacillus cereus was detected in three samples of cheese 41 , Bacillus cereusis a frequently isolated from food and food products, dairy products, it secretes toxins that can cause sickness and diarrhoea symptoms in humans. B. cereus is responsible for biofilm formation on food contact surfaces, such as stainless steel pipes, conveyor belts and storage tanks. It can also form floating or immersed biofilms, which can secrete a vast array of bacteriocins, metabolites, surfactants, proteases and lipases, in biofilms, which can affect qualities of food 42 . Motility by bacterial flagella confers access to suitable biofilm formation surfaces, and is required for biofilms to spread on non-colonised surfaces. However, B. cereus flagella have not been found to be directly involved in adhesion to glass surfaces, but can play a key role in biofilm formation via their motility 43 . B. cereus that contaminates both milk and milk products is based on the fact that usually contaminate milk during milking or storage on the farm, then gain entrance to dairy products from which they are prepared that depends on the effectiveness of hygienic measures applied during, handling, processing and distribution products of milk 44 .
Oliveira et al 45 evaluated multispecies biofilms formed on stainless steel (SS) due to the contaminating microbiota in raw milk and genetic diversity analysis indicated that Gammaproteo bacteria and Bacilli predominated in the biofilms, they have spoilage potential and they representatives of great importance. The biofilms can be formed on the surfaces of dairy processing equipment and are a potential source of product contamination. Pseudomonas spp. produce EPS huge amounts and are known to attach stainless steel surface and form biofilms. They can co-exist in biofilms with other pathogens to form multispecies biofilms, which make them more resistant and stable 46 . These biofilms can be accompanied by a distinct blue discolouration (pyocyanin) on fresh cheese produced by P.
Anoxybacillus flavithermus is Grampositive, thermophilic, and spore-forming organism that is facultatively anaerobic and non-pathogenic 48 . A. flavithermus spores are resistant to heat and their vegetative cells can grow at temperatures up to 65°C with a significant increase in bacterial adhesion on stainless steel surfaces in the presence of skimmed milk, and this indicator that milk positively influences these species' biofilm formation 49 . The commonest isolates that producing biofilm are thermophilic genera in the dairy industry as reported by Burgess et al 50 . It is essential that Biofilm-related effects in food industries as (pathogenicity, corrosion of metal surfaces, and alteration to organoleptic properties based on proteases or lipases secretion) are critically important. For example, in the dairy industry several structures and processes (pipelines, raw milk tanks, butter centrifuges, pasteurisers, packing tools, cheese tanks) can act as biofilm production surface substrates at different temperatures and involve several mixed cell species. Thus, to avoid contamination and to ensure food safety in the food industry, accurate methods to visualise biofilms in situ be set up 51 . For fighting biofilms 52 reported that two strategies in the industry of food: structural modification of surfaces or application of antibacterial or antibiofilm coatings 53 . Thus, several alternative products to classic disinfectants (chlorine, quaternary ammonium, etc.), such as, plantderived antimicrobials being the compounds that display more significant antimicrobial action in shorter action times as reported by EL-Hamshary et al 13 that ethanolic and ethyl acetate extracts of Tamarix nilotica plant showed antibacterial activity against B. cereus (S1) Staphylococcus aureus (S2); Bacillus paramycoides (S3); Staphylococcus aureus (S5); Serratia proteamaculan (S6); Serratia proteamaculan (S7) and Serratia proteamaculan (S9)) bacterial strains.
The aim of work is to study the prevalence of bacterial contamination in Domiaty and Hungarian cheeses collected from two big markets in Jeddah City. Identification of bacterial isolates by morphological characterization, biochemical test, biomerieux Vitek MS and molecular identification by 16S rRNA gene. The ability of bacterial isolates (29 bacterial isolates were tested for produce biofilm (16 Domiaty and 13 Hangarian cheese) using Tissue Culture Plate (TCP) quantitative technique.

Media preparation
Different media were used as nutrient agar (NA) 54 , MacConkey agar adjusting pH to 7.4 54 . All media during the present study were sterilized by autoclaving at 121 o C for 2hrs and used for bacterial growth experiments.

Collection of cheese samples
Domiaty and Hungarian cheeses were collected from two markets (M and P) in Jeddah city. Samples transported aseptically in ice container under refrigeration temperature 4°C to be tested immediately at the laboratory.

Isolation of bacterial isolates Preparation of samples
One gram from each cheese sample was taken from the upper surface and blended with 9 ml of sterile distilled water in falcon tube were prepared on serial dilution method from 10 -1 until 10 -7 and 100 microliter of each dilutions were spread on top of the nutrient agar (NA) medium then incubated at 37°C for 24 to 48 hrs.

Viable bacterial counts
This method was used to enumerate the total count of viable bacteria, bacterial colony were picked up after 24 to 48 hrs. on (NA) from each diluted cheese sample. Colonies were counted (total cell count) and the results were expressed as (C.F.U/ml) estimated on standard plate count (SPC) 55 .

Bacterial isolation and purification
Specific bacterial colonies were selected according to morphological study such as: color, size and margin, then isolated and purified by repeated streaking on the (NA) agar medium plate and incubated at 37°C for 24 to 48 hrs to obtain pure single colony.

Morphological characterization of bacterial isolates
Gram staining of isolates of bacterial was carried out using method as reported by Allan et al 56 .

Biochemical identification indol test
Indole test determines the ability to decomposing microorganism amino acid tryptophan to indole. Bacterial isolates from cheeses were grown on NB medium for 24 to 48 hrs. at 37°C before used. Indole urease medium of indole test was prepared and 5 ml was fill to all test tubes then transfer one ml from each bacterial isolate test tube, and uninoculated tube was kept as control. If tryptophan oxidized by bacteria, cherry red color was appeared on the top layer that indicated a positive result while if cherry red color wasn't appeared that indicated negative result 57 .

Catalase test
Catalase test facilitates to detect the presence of catalase enzyme. This enzyme produced by bacteria which use oxygen in respiration. Catalase enzyme break down hydrogen peroxide H 2 O 2 into water and hydrogen. Single colony from fresh bacterial isolates that grown on NA and transferred on clean glass slide then a drop of 30% [v/v] H 2 O 2 solution was placed on it. Appearance of bubbles indicated positive result (CAT+) while no bubbles mean negative result (CAT-) 58 .

Oxidase test
This test used to determination the presence of cytochrome enzyme oxidase in bacteria. The reagent used is a dye (TMPD) acts as an artificial electron accepter substituting the oxidase. Single colony from fresh bacterial isolates that grown on NA medium. Cotton swaps dipped in oxidase reagent (TMPD) then touched the colony of fresh selected isolates to test them. Blue-purple color appeared on filter paper mean oxidase positive, while yellow color mean oxidase negative 59 .

starch hydrolysis
This test examined the ability of isolate to produce a-amylase on medium containing starch as carbon source. The bacterial isolates were grown on starch nitrate agar medium at 37°C for 2 days. All plates were flooded with iodine solution for 3 minute appearance of clear zone around the growth indicated the starch hydrolysis while blue color mean no hydrolysis 60 .

Identification of bacterial isolates Identification by biomerieux VITEK® MS compact system
VITEK MS is an automated mass spectrometry microbial identification system that uses Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology (MALDI-TOF) has been shown to be both accurate in the identification of bacteria and rapid 61 . The methods as described by Westblade et al 62 .

Molecular identification of isolats based on 16S rRNA sequencing
Bacterial colonies isolated from cheese samples were molecularly identified using sequencing of the 16S rRNA. GeneJET Genomic DNA extraction kit used for extract genomic DNA according to the manufacturer's instructions. DNA extracted were amplified by polymerase chain reaction (PCR) using 16S rRNA universal primer pair (The forward primer 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3 and reverse primer 1492R 5'(TAC GGY TAC CTT GTT ACG ACT T)3') to amplify the 16s rRNA gene. 29 bacterial isolates were tested for produce biofilm formation (16 Domiaty and13 Hangarian cheese) using Tissue Culture Plate (TCP) quantitative technique then sequences compared with the available sequences against the 16S rRNA sequences database using NCBI's Blast N.(www. ncbi.nlm.nih.go).

Biofilm detection method Tissue culture plate (TCP)
Biofilm assay was performed based on growth and biofilm formation of bacteria in 96 well microtiter, Tissue culture plate (TCP) is considered as a standard test for the detection the production of biofilm. The overnight cultures grown in NB were diluted at 10 -3 and inoculated into six individual wells of a Tissue Culture Plate Method (150µl per well). Then the plates were incubated for 24 hrs. at 30 C. The ability of bacterial isolates (29 bacterial isolates were tested for produce biofilm (16 from Domiaty and 13 from Hangarian cheese) using Tissue Culture Plate (TCP)  RESUlTS AND DISCUSSION Collction of cheese sampels Eighteen samples of Domiaty and Hungarian cheeses were obtained from two big markets (M and P) in Jeddah City at a period between February to October 2018.

Isolation of bacterial isolates from cheese samples
Results in Table (1) showed the total bacterial counts (CFU/ml) from Domiaty cheese from two markets (M and P). The results indicated that the number of bacterial isolates were 0.1 x 10 5 , 8 x 10 5 and 1 x 10 5 CFU/ml respectively from 3 visits of M market. Six samples from 6 visits were collected from P market. The results revealed that the number of bacterial isolates were 4 x 10 6 , 0.4 x 10 6 , 6.5 x 10 3 , 1 x 10 3 , 0.1 x 10 3 and 0.1 x 10 3 CFU/ml respectively. Results in Table (2) showed the total bacterial counts (CFU/ml) from Hungarian cheese from M and P markets. The results indicated that the number of bacterial isolates were 1.5 x 10 5 , 1x 10 4 , 11 x 10 4 and 4 x10 6 CFU/ml respectively from 4 visits of M market. The results revealed also that the number of bacterial isolates were 0.18 x 10 4 , 3 x 10 6 , 22 x 10 6 , 6 x 10 6 and 5 x 10 4 CFU/ml respectively from 5 visits were obtained from P market.
Minimum (Min) bacterial count of Domiaty cheese from M market was 0.1 x 10 4 CFU/ ml, and Maximum (Max) was 8 x 10 4 CFU/ml. (Min) bacterial count of Domiaty cheese from P market  (4) shows morphological characteristics of bacterial isolates obtained from Hungarian cheese. Results revealed that (three of bacterial isolates Gram-positive bacilli, 1 of bacterial isolates Gram-negative bacilli and 6 of bacterial isolates Gram-positive coccus). These isolates represented 38.46%, 7.69% and 53.84% respectively.

Biochemical test of bacterial isolates from cheeses
Sixteen bacterial isolates of Domiaty cheese and thirteen bacterial isolates of Hungary cheese were tested for indole, catalase, oxidase, gelatin liquefaction and starch hydrolysis. Results of biochemical test of bacterial isolates from Domiaty and Hungarian cheese showed at Table (5).

Molecular identification of isolates based on 16S rRNA gene
Sequence analysis of the 16S rRNA gene has been measured fast and precise technique to recognize the phylogenetic position of bacteria. Then sequences were submitted to GenBank at NCBI web site (www.ncbi.nlm.nih.gov) under accession numbers: MK757978 (Raoultilla terrigena (D15-1)), MK757979 (Bacillus cereus (D16-1)), MK757980 (Enterococcus faecalis (H10-2)), MK757982 (Enterococcus fiscalism (H11-1) ), MK757981 (Serratia liquefactions(H13-1)), MK757984 (Anoxybacillus flavithermus (H17-1)). Results of Blast search for DNA sequence in NCBI Genbank were shown in Table (  pathogenicity, and alteration to organoleptic properties based on of proteases or lipases secretion are very important. For example, in the dairy industry several structures and processes (pipelines, raw milk tanks, butter centrifuges, pasteurisers, packing tools, cheese tanks,) can act as surface substrates for form biofilm at different temperatures and involve several mixed colonising species. Thus, it is essential that accurate methods to visualize biofilms in situ be set up to avoid contamination and to ensure food safety in the food industry 51 .
Results in this study indicated that Enterococcus faecium (H11-1) bacterial strains isolated from Hungarian cheese produced strong biofilm and Enterococcus faecalis (H10-2) that form moderate biofilm. Different lactic acid bacterial species were isolated and identified from Domiaty cheese, (Lactobacillus delbrueckii subsp. bulgaricus, L. casei, Lactococcus lactis subsp. lactis )as reported by (Fahmy and Youssef), L. farciminis, L. alimentarius, E. faecium, Enterococcus faecalis 12,67 reported that, the high rate of contamination of the examined Journal of Pure and Applied Microbiology cheese samples with Enterobacteriaceae is indicative for direct or indirect fecal pollution of milk used, neglecting of hygienic measures during production and handling and possible presence of enteric pathogens. Mohamed and Huang 68 reported that E. faecium and E. facials isolated from cheese and can be form biofilm. Kristich et al 69 reported that E. facials formed complex biofilm. But E. facials cannot form biofilm because some types of cheeses and curd cheeses incapable of biofilm formations. One of the reasons why Enterococcus spp. isolated from cheeses did not form biofilm could be due to the presence of sodium chloride in cheese (up to 4%) and a higher acidity of curd cheese (up to 70 SH) 70 .
This study revealed that Anoxybacillus flavithermus (H17-2) bacterial strain isolated from Hungarian cheese produced high biofilm (strong biofilm). Anoxybacillus flavithermus is Gram-positive, thermophilic, and spore-forming organism that is non-pathogenic Strejc et al. It is a the rmophilic bacterium that is able to survive at temperatures ranging from 55 to 60°C, Khalil et al 71 and Goh et al 72 reported that A. flavithermus isolated from diary processing plant., and also the commonest biofilm-forming isolates are thermophilic genera in the dairy industry 43  From our study, contaminant bacteria (Bacillus cereus (D16-1) were isolated from Domiaty cheese and produced moderate biofilm formation. Bacillus cereus group may be present in a wide variety of dairy products such as milk, pasteurized milk, powdered milk, cheeses and fermented milk 74, 75 reported that Bacillus cereus contaminated the requeijao curd cheeses. Also, isolated from feta cheese 76 . Bacillus cereus is a Gram-positive anaerobic or facultative anaerobic spore-forming bacterium that can grow in various environments at wide-ranging temperatures (4°C-50°C). It is resistant to chemicals, heat treatment, and radiation 40 . B. cereus is a frequently isolated from food and food products, such as dairy products. It secretes toxins that can cause sickness and diarrhoea symptoms in humans. B. cereus is responsible for biofilm formation on food contact surfaces, such as stainless-steel pipes, conveyor belts and storage tanks. It can also form floating or immersed biofilms, which can secrete a vast array of bacteriocins, metabolites, surfactants, as well as enzymes, such as proteases and lipases, in biofilms, which can affect food sensorial qualities 42 . Motility by bacterial flagella confers access to suitable biofilm formation surfaces, and is required for biofilms to spread on non-colonised surfaces. However, B. cereus flagella have not been found to be directly involved in adhesion to glass surfaces,  83 reported that most S. thermophilus strains are poor biofilm producers, mostly because they have lost these traits, consistent with their adaptation to the milk environment and selection as starters for dairy fermentations.
Miao et al 52 reported that two strategies in the industry of food: structural modification of surfaces or application of antibacterial or antibiofilm coatings 53 . Thus, several alternative products to classic disinfectants (chlorine, quaternary ammonium, etc.), such as, plantderived antimicrobials being the compounds that display more significant antimicrobial action in shorter action times as El-Hamshary et al 13 .