Molecular characterization and microbial resistance of different bacterial isolates in some dairy products

Waleed K. Alkhafaje¹, Zakia A. Olama¹ and Safaa M. Ali^2*

¹Botany and Microbiology Department, Faculty of Science, Alexandria University, ²Nucleic Acid Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological applications, Alexandria, Egypt

waleedalkhalid85@gmail.com, 0000-0002-8021-1603

zzoo5@hotmail.com, 0000-0002-0095-4029

safaa.mohamedali@yahoo.com, 0000-0001-9850-0174, corresponding

Abstract

Bacterial contamination of milk and dairy products is a common problem. In the last two years, the foodborne diseases caused by the intake of milk and dairy products have been mostly disturbed with Salmonella entertica, Listeria monocytogenes Escherichia coli 0157:H7 and Campylobacter jejune. The study aims to isolate multidrug resistance (MDR) bacteria in dairy products and study of the molecular characterization of that isolates. MDR bacteria were found in 30 out of the 131 bacterial isolates. The incidence of MDR bacterial isolates revealed the abundance of Staphylococcus sp. with 43.3%, Bacillus sp 16.7%, Salmonella 13.3%, E. coli 10 %, Enterococcus 6.7 % Psedoumonas 3.3 %, Shegella 3.3 % and Proteous 3.3 %. Molecular studies of genes presence or absence for class A contain TEM, CTX and BSHV, class B contain VIM, IMP, KPC and NDM, class C contain FOX and class D contain OXA-10, OXA-24 and OXA-58 were tested. NDM, TEM, CITM and OXA -10 genes were the most abundant the selected bacterial isolates. The results of this study indicate that cheese made from unpasteurized milk can pose a significant risk to consumers. Product manufacturing processes should be subject to health control-to-control pathogens. The novelty in this work depend on screening of gene responsible of the resistance from the bacteria isolated from dairy product using the molecular technique.

Keywords: Multidrug resistance, Gene, Bacteria, Milk, Dairy products

التوصیف الجزیئی والمقاومة المیکروبیة للعزلات البکتیریة المختلفة فی بعض منتجات الألبان

ولید خالد الخفاجى¹، زکیة أحمد علما¹ و صفاء مجمد على²

¹قسم النبات والمیکروبیولوجى، کلیة العلوم، جامعة الإسکندریة، ²قسم بحوث الأحماض النوویة، معهد الهندسة الوراثیة، مدینة الأبحاث العلمیة والتطبیقات التکنولوجیة، الإسکندریة، مصر

الخلاصة

یعد التلوث الجرثومی للحلیب ومنتجاته احدى اهم المشاکل شائعة الحدوث. فی العامین الماضیین، تحدث الأمراض التی تنتقل عن طریق الغذاء نتیجة تناول الحلیب ومنتجاته إلى اضطراب توزیع لبکتریا السالمونیلا انتتیریکاو اللیسیریا وایشیریشیا کولای وکمبیلاباکتر جیجونا. فکان الهدف الرئیسی من الدراسة هو عزل بکتیریا التی لها قدرة على مقاومة العدید من المضادات الحیویة التى عزلت من الحلیب ومنتجات الألبان ودراسة التوصیف الجزیئی لتلک العزلات. إن 30 عزلة بکتیریة من أصل 131 عزلة کانت مقاومة للعدید من المضادات الحیویة. کشفت الدراسة إن البکتریا من نوع الاستافیلوکوکس هی أکثر الأنواع الموجودة بین تلک العزلات بنسبة 43.3٪ ،16.7٪ من نوع الباسیلس، السالمونیلا کانت 13.3٪، الإشیریشیا کولاى کانت 10٪، الانتیروکوکاس 6.7٪ و 3.3٪ للسیدوموناس، الشیغیلة کانت 3.3٪ والبروتیوس کان 3.3٪. لقد تمت الدراسات الجزیئیة لوجود الجینات أو غیابها للفئة أ (TEM و CTX و BSHV) والفئة ب (VIM و IMP و KPC و NDM) والفئة ج (FOX) والفئة د (OXA-10 و OXA-24 و OXA-58). کانت جینات NDM و TEM و CITM و (OXA -10) الأکثر وفرة فی العزلات البکتیریة المختارة. تشیر نتائج هذه الدراسة إلى أن الجبن المصنوع من الحلیب غیر المبستر یمکن أن یشکل خطرا کبیرا على المستهلکین. یجب أن تخضع عملیات تصنیع منتجات الألبان إلى الرقابة الصحیة لمنع مسببات الأمراض. تعتمد الحداثة فی هذا العمل على فحص الجین المسؤول عن المقاومة من البکتیریا المعزولة من منتجات الألبان باستخدام تقنیة البیولوجیا الجزیئیة.

Introduction

The spoiling milk products worldwide is a huge economic problem. The microbial capacity and incidence of the bacterial pathogens in foods are indicators of the quality and safety of food. In addition, the education of the food handlers about the personal hygiene is importance from the food safety point of view (1). The highly nutritious nature of the dairy products makes them especially good media for the growth of microorganisms (2). The microbial contamination is one of the leading causes of the food spoilage worldwide (3). The contamination of food with microbes can occur at any stage of the food chain (4). The large number of diseases caused by foodborne pathogens with significant effects on economy and human health. The bacterial pathogens use a variety of different motility modes, including swimming, twitching, and swarming (1). Staphylococcus. aureus is commonly food born pathogen of the great importance of animal and human concern. It is responsible for contaminate dairy products, kariesh cheese and ice cream from the different sources during their production, processing and handling that make them unfit for human consumption or even a dangerous source of the infection among customers establishing a potential health hazard (5). Also, S. aureus known for its ability to secrete a lot of toxins to aid in host tissue infiltration and a acquire nutrients (6). Salmonella, Shigella, Listeria monocytogenes, Bacillus cereus, Campylobacter, Clostridium, Cronobacter, Escherichia (E.) coli, Staphylococcus (S.) aureus, Vibrio, Yersinia (Y.) enterocolitica, viruses such as hepatitis A and Noro viruses, and parasites Cyclospora (C.) cayetanensis, Toxoplasma (T.) gondii, and Trichin (7). The antimicrobial resistance (AMR): has the ability of kill microbes or limit their growth to grow in the presence of a drugs (8). The AMR complicates infection treatment and linked to increased mortality and morbidity. The emergence and spread of resistant and multidrug-resistant (MDR) bacteria has the enormous implications for worldwide healthcare delivery and the population health (9). The virulence functions encoded on the large extrachromosomal plasmids by pathogenic bacteria. These plasmids maintained at the low copy number to reduce the metabolic burden on their host (10). The widespread use of extended-spectrum cephalosporin creates a reservoir of resistant bacteria. Moreover, multi-resistance frequently associated with strains carrying (Extended-spectrum beta-lactamase) ESBLs, which could dramatically reduce the treatment options. The increasing number of Enterobacteriaceae with ESBLs that also contain (Metallo-β-lactamases) MBLs or AmpCs and other new mechanisms of resistance to fluoroquinolones or aminoglycosides indicate that recent increase of ESBLs -producing bacteria in Europe constitutes a complex problem (11).

ESBLs are worthy of the scientific community's attention over the past decades among the β-lactamases. ESBL's older and classical definition includes TEM-1, TEM-2, or SHV derivatives. ESBL divided into three main groups by the most recent definition. (I) ESBLA (class A ESBLs): CTX-M, SHV and TEM enzymes. (II) ESBLM (miscellaneous ESBLs) are sectioned into ESBLM-C (class C, plasmid mediated AmpC) and ESBLM-D (class D). (III) ESBLCARBA (ESBLsthat degrade carbapenems) are divided into ESBLCARBA-A, ESBLCARBA-B, and ESBLCARBA-D. More than 500 𝛽-lactamases reported to date produced by diverse bacteria. Beta-lactamases thought to be the most common resistance mechanism that contributes to widespread resistance among Gram-negative microbes also transmission of resistance occur between microorganisms (12). NDM-1 producing by E. coli infects the host by commonly invading sites like, urinary tract, blood, lungs, and wounds, leading to the urinary tract infections, septicemia, pulmonary infections, diarrhea, peritonitis, device-associated infections and soft tissue infections (13). The study's goal is to identify MDR bacteria from dairy products and examine their molecular characteristics.

Materials and method

Sample collection and analysis of microbiology

Dairy products: samples from different locations in Alexandria collected during 2018: white cheese, white cheese produced, old cheese and milk. The sample collected for further use in sterile containers. To evaluate the incidence of isolates of bacterial ESBL and CR isolated from the certain dairy products at the various locations in Alexandria, Egypt. 100 sample were collected that were distributed as Kareish cheese, Industrial white cheese, Old cheese, Roomy cheese and milk 40, 28, 15, 10 and 7% respectively.

Assessment of isolated bacteria's resistance prevalence

According to the modified Kirby-Bauer Disc Diffusion method, all the isolated bacteria subjected to the antibiotics resistance by using the disk diffusion method.

Microorganisms and molecular identification

The antibiotic resistant isolates subjected to phenotypic identification using the cultural characteristics in a trail to identify. Gram staining and analysing biochemistry. The region of 16S rRNA was amplified by using the universal primers F: AGAGTTTGATCMTGGCTCAG and R: TACGGYACCTTGTTACGACTT. The PCR reaction, was performed for 4 min at 95ºC followed by 40 cycles each of (Denaturation: 40 second at 94ºC, annealing: 50 second at 58ºC and extension: 50 second at 72ºC), followed by a supplementary 10min at 72ºC. Sequences of the 16S rRNA genes obtained from the NCBI database. Multiple alignments based on the most closely related sequences and the similarity levels carried out by using the BLAST program1. A phylogenetic tree reconstructed using the Mega 5 software.

Bacterial resistance determination using molecular techniques

The fresh bacterial cells used to extract DNA by using the GeneJET Genomic DNA Purification Kit. GEBRI kit removed plasmid from the bacteria's selected isolates.

Virulence gene detection using PCR assay

Eighteen primers used in this study; the genus used for screening of ESBL class A were TEM, CTX BETA and BSHV. ESBL class B carbapenemase encoding genus were CITM, VIM, IMP, KPC and NDM. ESBL class C encoding genus ACCM. ESBL class D encoding genus was OXA-10, OXA-55, OXA58, OXA 60 and OXA 69. Screening for coding sequence of genes DHAM, EBCM, MEC A, VAN A, and TOHO1 evaluated. PCR reaction achieved for 4 min at 95ºC followed by 35 cycles each of (Denaturation: 40 second at 94ºC, annealing: 50 second at 50-60ºC and extension: 50 second at 72ºC), followed by a supplementary 10min at 72ºC. After amplification by PCR assay, the products checked in 2% agarose gel electrophoresis. The genetic miscellany was determined as the experimental number of differentiation (Table 1).

Table 1: primers used in the work-study

Results

Assessment of isolated bacteria's resistance prevalence

The multiantibiotic resistance shown by the biggest promising isolates. Seven bacteria isolates were isolated from milk but show sensitive effect towards the antibiotic used for detection of MDR bacteria. All the selected MDR isolates were resistance to metronidazole (MTZ). Incidence of antibiotic sensitivity of multi-antibiotic resistance bacterial isolates (Table 2). Distribution of the MDR bacterial isolates were 14 isolates from Kariesh cheese (out of 52 bacterial isolates), 11 isolates from industrial white cheese (out of 19 bacterial isolates), 3 isolates from old cheese (out of 37 bacteria isolates) and 2 isolates from roomy cheese (out of 13 bacteria isolates) (Figure 1).

Table 2: Resistance prevalence of the bacterial isolates

Figure 1: MDR incidence distribution in dairy product samples under test.

Identification of the bacterial isolates

To identify the bacterial selected with the most promising MDR 30 out of 130 isolates. The most commonly isolated pathogens were evaluated for phenotypic characterization and molecular identification using 16s rDNA. The amplified fragment sequences of 1500 bp identified. A dominant tool for identifying and classifying prokaryotes was the sequences of the different types of the strains from the gene bank. GenBank deposited the sequence of the most promising isolates and had the accession. From all the examined samples were staphylococcus spp, Bacillus spp, Salmonella spp, E. coli, Enterococcus spp, Pseudomonas spp, Shigella spp and Proteous spp. with 43.3, 16.7, 13.3, 10, 6.7, 3.3, 3.3 and 3.3 % respectively. 23.1% out of the total isolates 130 were pathogens.

Resistance determination using molecular technique

The variation between the isolated MDR bacteria shown by plasmid extraction of the selected bacterial isolates. The results showed that isolates 2, 9, 16, 19, 20, 24, 25, 27, 29, 30, 39, 40 and 41 were highly antibiotic-resistant positive plasmid with high copy numbers (Figure 2).

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Figure 2: Agarose gel electrophoresis plasmid profiles of different bacterial isolates.

Detection of the virulence gene using PCR assay (Detection of β-lactamase gene class A, B, C and D)

Collected data of genes presence or absence for class A (TEM, CTX and BSHV) (Figure 3), class B (CITM, VIM, IMP, KPC and NDM) (Figure 4), class C (ACCM) (Figure 5), and class D (OXA-10, OXA-55, OXA-58 and OXA 69) (Figure 6). Screening of DHAM, EBCM, MEC, VAN and TOHA gene tested by using the selected bacterial isolates (Figure 7). The fragment size of amplified PCR product was calculated by using the software of Gel Documentation Analysis System (Alpha Imager TM 1220). In addition, the results of the present study showed that the average percentage of positive genes in all the selected MDR (30 isolates). Distribution of presence or absence of the 18 tested virulence genes among the MDR bacterial isolates (Figurer 8).

Figure 3: Agarose gel electrophoresis of the amplified PCR fragment for Class A, no. refers to the code of positive isolate.

Figure 4: Agarose gel electrophoresis of the amplified PCR fragment for Class B, no. refers to the code of positive isolate.

Figure 5: Agarose gel electrophoresis of the amplified PCR fragment for Class C (ACCM gene), no. refers to the code of positive isolate.

Figure 6: Agarose gel electrophoresis of the amplified PCR fragment for Class D, no. refers to the code of positive isolate.

Figure 7: Agarose gel electrophoresis of the amplified PCR fragment for (EBCM, MEC, DHAM, VAN AND TOHA gene), no. refers to the code of positive isolate.

Figure 8: Average percentage of positive genes in all the selected MDR (30 isolates).

Discussion

Studies previously conducted in different countries revealed a wide S. aureus diversity in milk and dairy products. The prevalence of S. aureus was 43.3% and it was differing than which carried out from Iran and Italy on different dairy products revealed a lower S. aureus percentage. Lower prevalence noted in studies which detected S. aureus with 10, 11.3, 5 and 9.1% respectively for the kariesh cheese (31). Greater incidence of S. aureus was 72, 50, 70, 93 and 68 % respectively for the kariesh cheese (32). Percentage of Bacillus spp. in our study was 16.7% which were isolated from the industrial white cheese and the kareish cheese where it was higher percentage in industrial white cheese. It was differed than the study by EFSA 2005 which appeared the incidence of B. cereus was highest in karish cheese 25 to 80% during all the seasons followed by koshary 45 to 70% and cornsnacks 20 to 50%. Percentage of Salmonella sp. in our study was 13.3% that were isolated from the industrial white cheese and the kareish cheese 10 and 3.3%. Relatively lower results were obtained by Abo Zeed (33,34) where Salmonella spp. could not be identified in the surveyed of the white cheese samples. Ghada and colleagues (35) identified two samples polluted with Salmonella spp.

In our study, the E. coli percentage was 10% isolated from roomy, kareish and industrial white cheese compared to other bacterial isolates. In study by Abdelrahman et al., (36) found that, the incidence of E. coli in Kariesh, and Domiati cheese were 37.1% and 2.8%, respectively. While, could not be detect in Tallaga cheese. Naj and Ghanbarpour (37) who isolated E. coli with percentage of 98.7 % from the domestic soft cheese. A common principal of the resistance markers in all varieties estimated and associated antimicrobials such as tetracycline, B-lactams, sulfonamide and quinolones were detected (38). An aggregate number of extended-spectrum β-lactamases (ESBLs) have been predictable in Enterobacteriaceae through the latest few years. SHV types of enzymes shown to carry the SHV-1 gene within the chromosome (39). NDM-1 producers were resistant to imipenem, meropenem, ertapenem, gentamicin, amikacin, tobramycin, and ciprofloxacin, whereas, isolates were susceptible to colistin (40). The high prevalence of tetracycline- and penicillin-resistant S. aureus, Salmonella and E. coli observed in the current study, is in agreement with earlier findings. Gram-negative bacteria mostly preset TEM, almost 90% of the resistance against ampicillin in Gram-negative bacteria are due to TEM encoded genes (41).

Conclusion

The results of this study indicate that cheese made from unpasteurized milk can pose a significant risk to consumers. This risk varies depending on the geographical location of the study area, the state of education of the population, the extent of attention to hygiene and the method of preparing and packaging dairy products. Product manufacturing processes should be subject to health control-to-control pathogens. Dairy markets monitored for pathogens. Reducing the surface area exposed to air reduces harmful microbial growth in dairy products.

Conflict of interest

No conflict.

Acknowledgments

The authors acknowledge City of Scientific Research and Technological Applications.