Research Article | Open Access
Ola IM El-Hamshary1 , Sarah K. Abdullah2 and NH Al-Twaty2
1Department of Microbial, Genetics National Research Centre, Cairo, Egypt.
2Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
J Pure Appl Microbiol. 2021;15(2):983-997 | Article Number: 6775
https://doi.org/10.22207/JPAM.15.2.57 | © The Author(s). 2021
Received: 17/11/2020 | Accepted: 22/05/2021 | Published: 01/06/2021
Abstract

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.

Keywords

Domiaty and Hungarian cheeses, S. liquefaciens, P. fluorescens, Anoxybacillus flavithermus, 16S rRNA, biofilm detection

Introduction

The food is the fuel of our life and it is a major concern for quality and safety1. 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 phosphorus2. 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 Papademas3. Domiaty cheese is one of the most popular varieties of cheese, if contaminated, it causes of foodborne illness. Cokal et al4 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 to5, 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 plant6, 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 risks8. Cheese contamination can occur with foodborne pathogens in several stages in cheese processing, as pastoralized milk, row milk, after pastoralized milk9. 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 illness10. 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. farciminisL. alimentarius as reported by El-Zayat et al12 and EL-Hamshary et al13 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) matrix14, 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 Flemming15 reported that extracellular polymeric substances (EPS) are biosynthetic polymers produced by micro-organisms 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 environment16. Bacterial EPS affect cell adhesion, microbial aggregates formation (biofilms, flocs, sludges and bio-granules), as reported by Comte et al17). 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 areas19. 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 be related specifically to food-borne pathogens in biofilms20.

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 al21 and Colagiorgi22.

The characteristics of attachment surface’s affect the production of mixed-species biofilm23, conditions of environment24, and involved bacterial cells25,26. Food matrix components27, in food processing environments also influence attachment of bacteria28; 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 aureus29,30 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 adhesion31.

Lafarge et al32 detected Serratia spp. bacterial strains in different sources as raw milk, in a milk-processing plant33, milk bulk tank as reported by Decimo34, and from internal surfaces of tankers of raw milk and reported that produce (heat-resistant 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 surfaces35,36, 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 al33. The presence of a single different strain may have a significant effect on the microbial dynamics in dairy products32.

Machado et al37 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 proteases38, and from milk as showed by Gaffer et al39.

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 cheese41, 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 food42. 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 motility43. 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 milk44.

Oliveira et al45 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 stable46. These biofilms can be accompanied by a distinct blue discolouration (pyocyanin) on fresh cheese produced by P. fluorescens47.

Anoxybacillus flavithermus is Gram-positive, thermophilic, and spore-forming organism that is facultatively anaerobic and non-pathogenic48. 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 formation49. The commonest isolates that producing biofilm are thermophilic genera in the dairy industry as reported by Burgess et al50. 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 up51. For fighting biofilms52 reported that two strategies in the industry of food: structural modification of surfaces or application of antibacterial or antibiofilm coatings53. Thus, several alternative products to classic disinfectants (chlorine, quaternary ammonium, etc.), such as, plant-derived antimicrobials being the compounds that display more significant antimicrobial action in shorter action times as reported by EL-Hamshary et al13 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.

Materials and Methods

Media preparation
Different media were used as nutrient agar (NA)54, MacConkey agar adjusting pH to 7.454. All media during the present study were sterilized by autoclaving at 121oC 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 al56.

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 result57.

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 H2O2 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] H2O2 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 negative59.

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 hydrolysis60.

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 rapid61. The methods as described by Westblade et al62.

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) quantitative technique as described by Mathur
et al63.

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 105, 8 x 105 and 1 x 105 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 106, 0.4 x 106, 6.5 x 103, 1 x 103, 0.1 x 103 and 0.1 x 103 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 105, 1x 104, 11 x 104 and 4 x106 CFU/ml respectively from 4 visits of M market. The results revealed also that the number of bacterial isolates were 0.18 x 104, 3 x 106, 22 x 106, 6 x 106 and 5 x 104 CFU/ml respectively from 5 visits were obtained from P market.

Table (1):
Total bacterial count (CFU/ml) of Domiaty cheese.

Number of visits
CFU/ml
V1 (M)
0.1 x 10 5
V2 (M)
8 x 10 5
V3 (M)
1 x 10 5
V4 (P)
4 x10 6
V5 (P)
0.4 x10 6
 V6 (P)
6.5 x 103
V7 (P)
1 x103
V8 (P)
0.1 x103
V9 (P)
0.1 x103

Minimum (Min) bacterial count of Domiaty cheese from M market was 0.1 x 104 CFU/ml, and Maximum (Max) was 8 x 104 CFU/ml. (Min) bacterial count of Domiaty cheese from P market was 0.1 x102 CFU/ml and (Max) was 8 x 104 CFU/ml. This result is lower than the similar studies although64, collected Domiaty cheese from Cairo and Giza, results indicated the total bacterial count per gram CFU/g. At Cairo, (Min) bacterial count of Domiaty cheese was 9×102 CFU/g and (Max) bacterial count was 3×106. From Giza Minimum (Min) bacterial count of Domiaty cheese was 7×102 CFU/g and Maximum (Max) bacterial count was 2×108. Hungarian cheese obtained from M market and results indicated that CFU/ml were 1.5 x 104, 1x 103, 11 x 103 and 4 x105 obtained respectively while from P market, samples of Hungarian cheese obtained and results indicated that CFU/ml were 0.18 x103, 3 x105, 22 x105, 1 x102, 6 x105 and 5 x105 respectively. (Minimum) bacterial count of Hungarian cheese from M market was 1 x103 CFU/ml and (Maximum) was 4 x 105 CFU/ml. whereas from P market (Min) was 1 x102 CFU/ml and (Max) was 22 x105 CFU/ml. These results are similar and higher than in bacterial count  to that reported by Alper and Nesrin65, that indicated the total bacterial count of cheeses isolated from Turkey were 5.2 x10 4 and 5.68 x 1011 CFU/g.

Table (2):
Total bacterial count (CFU/ml) of Hungarian cheese.

Number of visits
CFU/ml
V1 (M)
1.5 x 10 5
V2 (M)
1x 10 4
V3 (M)
11 x 10 4
V4 (M)
4 x10 6
V5 (P)
0.18 x10 4
 V6 (P)
3 x 106
V7 (P)
22 x 106
V8 (P)
6 x 106
V9 (P)
5 x 104

Morphological Characterization of bacterial isolates
Morphological characteristics of isolates from Domiaty and Hungarian cheeses were summarized and presented in Tables (3 and 4) respectively. Results in Table (3) shows morphological characteristics of bacterial isolates obtained from Domiaty cheese. Results showed that 6 of bacterial isolates Gram-positive bacilli and 8 bacterial isolates Gram-negative bacilli. Result in Table (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.

Table (3):
Morphological characterization of bacterial isolates from Domiaty cheese.

Number of isolates Cell shape Gram stain Mackonckyagar Morphological characterization
Shape Margin Color size
D6-1 Bacilli + Circular Entire Cream Big
D6-2 Bacilli + Circular Entire Cream Small
D11-1 Bacilli + Circular Entire Cream Big
D11-2 Bacilli + Circular Entire Cream Small
D12-1 Bacilli + Circular Entire White Small
D12-2 Bacilli + Circular Entire White Small
D13-1 Bacilli + Circular Entire Cream Small
D13-2 Bacilli + Circular Entire Cream Small
D14-1 Bacilli + Circular Entire Cream Big
D14-2 Bacilli + Circular Entire White Medium
D15-1 Bacilli + Circular Entire Cream Big
D15-2 Bacilli + Circular Entire White Big
D16-1 Bacilli + Irregular Entire White Big
D17-1 Bacilli + Irregular Entire White Big

Table (4):
Morphological characterization of bacterial isolates from Hungarian cheese.

Number of isolates Cell shape Gram stain Mackonckyagar Morphological characterization
Shape Margin Color size
H6-1 Coccus + Circular Entire White Small
H6-2 Coccus + Circular Entire Cream Big
H10-1 Coccus + Circular Entire Cream Small
H10-2 Coccus + Circular Entire Cream Medium
H11-1 Coccus + Circular Entire White Small
H11-2 Coccus + Circular Entire White Medium
H13-1 Bacilli + Circular Entire White Small
H14-1 Strepto- coccus + Circular Entire Cream Small
H15-1 Bacilli + Circular Entire White Small
H16-1 Bacilli + Circular Entire White Small
H17-1 Bacilli + Circular Entire White Small
H17-2 Bacilli + Circular Entire White Small

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).

Table (5):
Biochemical test of bacterial isolates from Domiaty and Hungarian cheese.

Number of isolates
Indole test
Catalase test
Oxidase test
Starch hydrolysis
Gelatin hydrolysis
D6-1
+
+
+
D6-2
+
+
+
D10-1
D10-2
D11-1
+
D11-2
+
D12-1
+
D12-2
+
D13-1
+
+
+
D13-2
+
+
+
D14-1
+
+
+
D14-2
+
+
D15-1
+
D15-2
+
D16-1
+
+
D17-1
+
+
H4-1
H6-1
+
H6-2
+
H10-1
H10-2
H11-1
H11-2
H13-1
+
+
+
H14-1
H15-1
+
+
+
H16-1
+
H17-1
+
H17-2
+

Identification bacterial isolates
Identification by biomeriex Vitek MS compact system
Results of identification bacteria isolates by biomeriex Vitek MS compact system were shown in (Table 6). Six of bacterial isolates from Domiaty cheese were identified as (5 Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2) and one Pseudomonas fluorescens(D14-2))strains. Results showed that 3 isolates of bacteria were identified as (one Enterococcus faecium (H11-2), one Serratia liquefaciens (H15-1) and one Streptococcus salivarius spp. Thermophilus (H14-1)) strains isolated from Hungarian cheese.

Table (6):
Identification bacterial genus/species isolated from Domiaty and Hungary cheeses by Vitec MS.

Types of cheese
Bacterial Genus/Species
Domiaty cheese
Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2) Pseudomonas fluorescens(D14-2)
Hungarian cheese
Enterococcus faecium (H11-2)
Serratia liquefaciens(H15-1)
Streptococcus salivariusspp. thermophilus (H14-1)

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 (7).

Table (7):
Results of Blast search for DNA sequence in NCBI Genbank.

Isolates
Accession No.
Raoultillaterrigena (D15-1)
MK757978
Bacillus cereus (D16-1)
MK757979
Enterococcus faecalis (H10-2)
MK757980
Enterococcus fiscalism (H11-1)
MK757982
Serratia liquefactions (H13-1)
MK757981
Anoxybacillusflavithermus (H17-1)
MK757984

Biofilm detection method
In food industries, the effects related biofilm as corrosion of metal surfaces, 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 industry51.

Table (8):
Biofilm formation by Tissue Culture Plate (TCP) of Domiaty cheese isolates.

Number of isolates
(OD570 nm)
Standard
Biofilm formation
Adherence
S. liquefaciens (D6-1)
0.142
(0.12–0.24)
Moderate
Medium
D6-2
0.173
(0.12–0.24)
Moderate
Medium
D10-1
0.236
(0.12–0.24)
Moderate
Medium
D10-2
0.210
(0.12–0.24)
Moderate
Medium
D11-1
0.186
(0.12–0.24)
Moderate
Medium
D11-2
0.405
<0.24
High
Strong
D12-1
0.178
(0.12–0.24)
Moderate
Medium
D12-2
0.159
(0.12–0.24)
Moderate
Medium
D13-1
0.201
(0.12–0.24)
Moderate
Medium
D13-2
0.229
(0.12–0.24)
Moderate
Medium
D14-1
0.139
(0.12–0.24)
Moderate
Medium
D14-2
0.216
(0.12–0.24)
Moderate
Medium
D15-1
0.330
<0.24
High
Strong
D15-2
0.201
(0.12–0.24)
Moderate
Medium
D16-1
0.127
(0.12–0.24)
Moderate
Medium
D17-1
0.133
(0.12–0.24)
Moderate
Medium

In this study a total of 29 bacterial isolates were tested for produce biofilm formation (16 Domiaty and 13 Hangarian cheese)using Tissue Culture Plate (TCP) quantitative technique. All isolates were screened for their ability to form biofilm production by TCP that measured by using Micro-plate Reader at (OD570 nm) and considered zero (0.24) according to TCP
method66. Results of biofilm production of isolates from Domiaty cheese using method of TCP showed that 87.5% (14/16) were considered moderate biofilm formation as shown in (Table 8). Results indicated also that isolates (D11-2 and D15-1) OD570 nm were (0.405 and 0.330) respectively which considered high biofilm formation, were strong biofilm adherence. Results of biofilm production from Hungarian cheese revealed that 53.5% (7/13) were considered moderate biofilm formation as shown in (Table 9). Results showed also that 46.1% (6/13) (H6-1, H6-2, H11-1, H11-2, H13-1 and H17-2) OD570 nm were (0.303, 0.299, 0.307, 0.262, 0.242 and 0.362) respectively that considered high biofilm production (strong biofilm adherence).

Table (9):
Biofilm formation by Tissue Culture Plate (TCP) at OD570 nm of Hungary cheeseisolates.

Number of isolates
(OD570 nm)
Standard
Biofilm formation
Adherence
H4-1
0.220
(0.12–0.24)
Moderate
Medium
H6-1
0.303
< 0.24
High
Strong
H6-2
0.299
< 0.24
High
Strong
H10-1
0.236
(0.12–0.24)
Moderate
Medium
H10-2
0.220
(0.12–0.24)
Moderate
Medium
H11-1
0.307
< 0.24
High
Strong
H11-2
0.262
< 0.24
High
Strong
H13-1
0.140
 (0.12–0.24)
Moderate
Medium
H14-1
0.232
(0.12–0.24)
Moderate
Medium
H15-1
0.147
(0.12–0.24)
Moderate
Medium
H16-1
0.076
(0.05–0.12)
Weak
Weakly
H17-1
0.141
(0.12–0.24)
Moderate
Medium
H17-2
0.362
< 0.24
High
Strong

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. bulgaricusL. casei, Lactococcus lactis subsp. lactis )as reported by (Fahmy and Youssef), L. farciminisL. alimentariusE. faecium,  Enterococcus faecalis12, 67 reported that, the high rate of contamination of the examined 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 Huang68 reported that E. faecium and E. facials isolated from cheese and can be form biofilm. Kristich  et al69 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 al71 and Goh et al72 reported that  A. flavithermus isolated from diary processing plant., and also the commonest biofilm-forming isolates are thermophilic genera in the dairy industry43.  A. flavithermus spores are very heat-resistant 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. This indicates that milk positively influences these species’ biofilm formation Sadiq et al49 and Dai et al73 reported that A. flavithermus isolated from water and formed biofilm.

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 milk74,75 reported that Bacillus cereus contaminated the requeijao curd cheeses. Also, isolated from feta cheese76. 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 radiation40. 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 qualities42. 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 motility43. B. cereus and P. aeruginosa showed the highest biofilm formation77.

In our study, Pseudomonas florescence isolated from Domiaty cheese, and results agreement with78-82.

From this study, Serratia liquefaciens (H13-1) detected in (Domiaty and Hungarian) cheeses and produced moderate biofilm formation, this  results similar to Couvigny et al83 who reported that Serratia odoriferawas isolated from Italian cheeses
and Morales et al84 detected Serratia spp. in milk and cheeses. Serratia liquefaciens is a spoilage microorganism of relevance in the dairy industry because it is psychrotrophic, able to form biofilm, and produces thermoresistant proteases and lipases Rodrigues et al. and from milk39.

Bacterial strain Raoultilla terrigena
(D15-1) or Klebsiella terrigena obtained from Domiaty cheese that produce strong biofilm formation. These results similar to the results of Kongo and Gomes85 who reported that Klebsiella terrigena and  K. ornithinolytica strains isolated from cheddar cheese. Ogbolu et al86 reported bacterial contamination of cheeses by Klebsiella species. In our study, Streptococcus thermophilus isolated from Hungarian cheese and had mediate biofilm formation. Our results agreement with Bassi et al87 who reported mediates biofilm formation in dairy environments. Also, Couvigny et al83 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.

CONCLUSION

Results of identification bacteria isolates by biomeriex Vitek MS compact system indicated that Six of bacterial isolates from Domiaty cheese were identified as (5 Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2) and one Pseudomonas fluorescens(D14-2)) strains. Results showed that 3 isolates of bacteria were identified as (one Enterococcus faecium (H11-2), one Serratia liquefaciens(H15-1) and one Streptococcus salivarius spp.Thermophilus (H14-1)) strains isolated from Hungarian cheese. Selected isolates were identified by16 rRNA sequencing as (Raoultilla terrigena(D15-1)), (Bacillus cereus (D16-1)), (Enterococcus faecalis (H10-2)), (Enterococcus fiscalism (H11-1)),  (Serratia liquefactions (H13-1)), (Anoxybacillus flavithermus(H17-1). A total of 29 bacterial isolates were tested for produce biofilm formation (16 Domiaty and13 Hangarian cheese) using Tissue Culture Plate (TCP) quantitative technique. Results of biofilm production of isolates from Domiaty cheese showed that 87.5% (14/16) were considered moderate biofilm formation. Results indicated also that isolates (D11-2 and D15-1) OD570 nm were (0.405 and 0.330) respectively which considered high biofilm formation, were strong biofilm adherence. Results of biofilm production from Hungarian cheese revealed that 53.5% (7/13) were considered moderate biofilm formation.  Results showed also that 46.1% (6/13) (H6-1, H6-2, H11-1, H11-2, H13-1 and H17-2) OD570 nm were (0.303, 0.299, 0.307, 0.262, 0.242 and 0.362) respectively that considered high biofilm production (strong biofilm adherence).

Miao et al52 reported that two strategies in the industry of food: structural modification of surfaces or application of antibacterial or antibiofilm coatings53. Thus, several alternative products to classic disinfectants (chlorine, quaternary ammonium, etc.), such as, plant-derived antimicrobials being the compounds that display more significant antimicrobial action in shorter action times as El-Hamshary et al13. reported 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.

Declarations

ACKNOWLEDGMENTS
The authors are grateful to Dr. AYA Saeed for English editing of the manuscript.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
None.

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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