MOLECULAR CHARACTERIZATION OF ANTIBIOTICS RESISTANCE GENES OF ENTEROCOCCI ISOLATED FROM RAW MILK IN ASSIUT CITY

Enterococcus species are Gram-positive cocci that are characterized by being catalase-negative, facultative anaerobic bacteria, and non-spore forming. Enteroccoi is often isolated from environmental and animal sources and inhabits the human gastrointestinal tract. Enterococcus species, which gets its resources from the dairy industry, animals, and people, is one of the most abundant lactic acid bacteria in raw milk. The aim of the present study is to detect antibiotic resistance of Enterococci isolated from raw milk by phenotypic and genotypic methods. Vitek 2 Compact System was used to identify the samples and assess their antimicrobial susceptibility. Following that, drug resistance genes ( ermB , aph (3')-IIIa , and TetM ) and one virulence gene ( esp ) were molecularly detected by PCR. Twenty isolates of Enterococci were phenotypically identified by routine laboratory examination and Vitek2. High rates of antibiotic resistance were found to erythromycin and tetracycline with percentages of 65% and 35%, respectively. The presence of tetM and ermB in milk isolates was found to be 100%, similarly. No detection of aph (3′)IIIa was found in milk isolates. Esp was detected only in 5% of isolated samples. The prevalence of Enterococci among studied samples was 11.8% (20/170). The fact that Enterococci were resistant to erythromycin and tetracycline in our study was noteworthy since these bacteria could potentially be transmitted to people by drinking milk that has not been properly treated.


INTRODUCTION
Enterococci were first discovered in human flora in 1899, but it wasn't known until the end of the nineteenth century when two different teams Thiercelin, and MacCallum & Hastings, described human enterococcal infections in detail (Thiercelin, 1899;MacCallum and Hastings, 1899).
Enterococcus are Gram-positive cocci which are spherical or ovoid and are lactic acid bacteria (LAB).They vary in length )0.6-2.0 μm( and width )0.6-2.5 μm(.They characteristically form short chains and cluster together (Langella, 2018).The range of body temperatures between 35 and 37°C is ideal for the proliferation of Enterococcus species in both humans and animals (Ch'ng et al., 2019).
Additionally, Enterococci are resistant to drying and can survive in unfavorable settings like those seen in a hospital environment (Ch'ng et al., 2019).Lactic acid bacteria are found in raw bovine milk's core, mesophilic/ psychrotrophic microbiota (Quigley et al., 2013).
Faecal contamination does not seem to be a major factor in Enterococci entry into the dairy production chain, in spite of their connection to the intestinal microbiotas of humans and dairy animals.The main source of raw milk contamination comes from the milking machine (Kagkli et al., 2007).
Mastitis can potentially be a source of Enterococci that are multi-drug resistant (Wu et al., 2016).Seasonal variations in enterococcal variety in raw milk may also be influenced by the time of year (McAuley et al., 2015).
Contagious mastitis is primarily caused by Streptococci.One of the environmental causes of mastitis is Enterococci.Enterococci can be transmitted from the inflamed udder to humans is another matter of concern.(Różańska et al., 2019).
Both the host's immune system's resistance to infection and teat contact with the enterococcal bacterium can result in mastitis.Bovine mastitis can be caused by E. faecium and E. faecalis (Montironi et al., 2020).
Several Enterococci virulence factors, mainly in E. faecalis and E. faecium, have been discovered (Homayouni et al., 2018).The enterococcal virulence factors reported so far have been grouped into two categories: surface proteins that encourage colonisation of the host and the secreted chemicals that cause tissue damage to the host (Chajęcka-Wierzchowska et al., 2017).
The virulence factors that encourage adhesion (all surface proteins) include the aggregation substance (AS), the collagen binding proteins Accessory colonisation factor (ACE in E. faecalis and ACm in E. faecium), endocarditis specific antigen (Efa A) and enterococcal surface protein (Esp) (Chajęcka-Wierzchowska et al., 2017).
The resistance of Enterococci to some antibiotic kinds is inherent, while resistance to others has been acquired.It is also important to note that E. faecalis and E. faecium make up the majority of the resistant Enterococci.(Růžičková et al., 2020).
The ermB gene, which is found in several transposons and plasmids in species of the Enterococcus, Streptococcus, Clostridium, and Staphylococcus genera, is the most widely distributed gene that confers resistance to macrolides in Enterococci (Torres et al., 2018).
Tetracycline resistance can also develop through a mechanism that prevents the drug from binding to the ribosome.Tetracycline activity is prevented by resistance genes like tetM and others on the ribosome.(Blair et al., 2015).On mobile elements, resistance genes for AGmodifying enzymes are frequently discovered.The development of AGmodifying enzymes such as acetyltransferases (AACs), phosphotransferases (APHs), and nucleotidyl transferases (ANTs) is the most frequent route of resistance to AGs in clinical isolates (Potron et al., 2015).

MATERIALS AND METHODS
Ethical statement.According to the World Medical Association's code of ethics (Declaration of Helsinki), the study was approved by the ethical committee of Assiut University's faculty of medicine.The number is 17101901.
Collection of samples: 170 raw milk samples were collected from Assiut University farm, farms from rural areas and milk laboratories in Assiut City.Samples were collected under sterile conditions.
Preparation of raw milk samples: For isolation of Enterococci from milk, the samples were centrifuged for 10 minutes at 5000 rpm with the supernatant being discarded afterward.A loopful from prepared sediments was streaked onto the surface of the blood agar plate and then incubated at 37°C for 18-24 hrs.(Abd El Tawab et al., 2019).

Culture on solid media:
 Blood agar: Enterococci emerged as nonhemolytic smooth, white-colored colonies with clean edges (Murray, 1999).A category interpretation will be reported along with a MIC, according to the interpretations defined by CLSI® 2021.

Molecular detection of resistant genes and virulence genes:
The primers used for the detection of both resistant genes and virulence genes are shown in the following table (1).
Table 1: Primers used in the detection of antibiotics resistance genes and virulence genes.
PCR assays: PCR was performed in a 25 μL reaction mixture containing 1 μL each of both forward and reverse specific primer pairs, 12.5 μL of PCR master mix (Thermo Fisher Scientific, United States)), 5.5 μL of nuclease-free water, and 5.0 μL of DNA template.
Two multiplex PCR were done for each of two gene sets (ermB and esp) and (aph(3′)IIIa and TetM).
ErmB and esp genes: Initial denaturation at 95°C for 5 minutes, 40 cycles each cycle consisting of denaturation at 95°C for 40 seconds, annealing at 55°C for 50 seconds, extension at 72°C for 50 seconds and final extension at 72°C for 5 minutes.

TetM and aph(3')-IIIa genes:
Initial denaturation at 95°C for 5 minutes, 40 cycles each cycle consisting of denaturation at 95°C for 40 seconds, annealing at 50°C for 50 seconds, extension at 72°C for 50 seconds and final extension at 72°C for 5 minutes.
Detection of the amplified product.The resulting PCR amplicons were examined on a 1.5% agarose gel, stained with ethidium bromide, and observed for two hours under UV illumination at 80 volts.
Statistical analysis.Categorical variables were described by number and percent (N, %), All analyses were performed with the IBM SPSS 26.0 software.

RESULTS
The prevalence of Enterococci isolates from raw milk was 11.8%.The most species isolated from milk were E. feacuim accounting for 30%, E.feacalis accounting for 25%, E. durans 30% and E.hiare15%.

Antimicrobial susceptibility test:
Tetracycline had the highest resistance rate with a percentage of 35%, followed by Quinuprisia/Dalfopristin with a percentage of 30%.Erythromycin resistance was observed in milk samples at a rate of 65%.Vancomycin-resistant Enterococci were only 10%.Linezolid-Resistant Enterococci was only 5% in the present study.About 10%, and 5% had resistance to benzylpenicillin and ampicillin, respectively.No resistance was detected at high levels of streptomycin, nitrofurantoin, or tigecycline as in Table (2).In milk samples, only 2 strains were found to be multidrug-resistant, and the other strains were found to be mostly sensitive, with a prevalence of 90%.In the current study, 90% of the Enterococci strains isolated were sensitive strains with resistance to one or two antibiotics.Only two strains were multidrug resistant; one was resistant to 4 groups of antibiotics (macrolides, streptogramin, glycopeptide and tetracycline), and the other was resistant to 5 groups of antibiotics (β-lactams, aminoglycosides, fluoroquinolones, glycopeptide and tetracycline).The multidrug-resistant strains isolated were found to be E.faecium.In contrast, Gołaś-Prądzyńska et al. (2022) reported that Multidrug-Resistant Enterococci (mostly E.faecium and E.feacalis) were resistant to at least of three groups of antibiotics which constituted 19.7%.

Molecular
In the present work, the resistant genes were present in milk samples with a high prevalence rate.The ermB and tetM resistant genes were 100% detected in our study, but the aph(3′)IIIa resistant gene was not detected in milk samples.Gołaś-Prądzyńska et al. (2022) mentioned that the prevalence of tetM in milk was 94.7%, which is almost in agreement with our data.In contrast, only a small amount of ermB was found in their research.In addition, Hammad et al. (2022) detected the prevalence of ermB and tetM in their study at 4.1% and 16.6%, respectively.Morandi et al. (2015) reported that the ermBresistant gene was not found in their study, but the tetM gene was found in 53% of the raw milk samples.Based on a study by Kang et al. (2021), the prevalence of tetM and ermB were 36.3% and 71.4%, respectively.Also, Bag et al. (2022) demonstrated the prevalence of resistant genes: tetM and aph(3′)IIIa were 50% and 12.5%, respectively.According to Đorđević et al. (2022), the prevalence of ermB, tetM, and aph (3′) IIIa was 19.2%, 8.5%, and 17%, respectively, in Serbia.
Interestingly, 35% of erythromycinsusceptible strains carried the ermB resistance gene, while 65% of erythromycinresistant strains carried ermB isolated from milk samples.The lack of correlation between phenotype and genotype in the ermB-resistant gene was discovered by Demirgül and Tuncer (2017) and Đorđević et al. (2022), and this could be due to the absence of gene expression, which is known as a "silent gene".In our study, a silent gene was also found to be present in the tetracycline-resistant gene: 35% of the tetracycline-resistant strains and 65% of the tetracycline-susceptible strains both included the tetM-resistant gene.The presence of the tetM silent gene was documented by Đorđević et al. (2022), who reported that one case was susceptible to tetracycline despite carrying the tetM-resistant gene.Interestingly, Morandi et al. (2015) also reported that 37% of tetracycline-susceptible strains carried a tetM resistance gene.Silent genes are known to exist, and their presence may be explained by inactive gene products or by low levels, or downregulation, of gene expression.
Esp, an enterococcal surface protein is connected to the growth of biofilms.Vancomycin, esp, and multiple antibiotic resistance are all related in E. faecium strains, as has been proven by Ochoa et al. (2013).In the current study, 5% of the milk samples had the virulence gene (esp).The prevalence of the esp virulence gene in dairy products was determined to be 4.1% by Hammad et al. (2015), which is consistent with our results.
Only two strains in milk samples isolates were found multi-drug resistant.The two multi-drug resistant strains were found to carry two resistant genes (ermB and tetM).

CONCLUSION
Our present study revealed that the prevalence of Enterococci isolates in milk was 11.8%.Two strains were found to be Multidrug-Resistant were isolated in milk samples.ErmB and TetM were found in milk with high percentages.Some samples of milk that were susceptible to tetracycline and erythromycin were found to carry their specific resistant genes which is called silent gene.The fact that Enterococci were resistant to erythromycin and tetracycline in our study was noteworthy since these bacteria could potentially be transmitted to people by drinking milk that has not been properly treated.Abdeltawab, A.A.;Mohamed, R.S. and Kotb, M.A. (2019) Blair, J.;Webber, M.A.;Baylay, A.J.;Ogbolu, D.O. and Piddock, L.J. (2015): Molecular mechanisms of antibiotic resistance.Nature Reviews Microbiology, 13(1), 42-51. Bouymajane, A.;Filali, F.R.;Oulghazi, S.;Ed-Dra, A.;Benhallam, F.;El Allaoui, A. and Moumni, M. (2018)

Table 2 :
Antimicrobial susceptibility test of Enterococci isolated from milk by vitek2:

Table 3 :
Coexistence of resistant genes and esp in milk samples.

Table 4 :
Relation between phenotype and genotype of antibiotics resistance genes of Enterococci isolated from milk samples.