Prevalence of antimicrobial-resistant Streptococcus species among respiratory patients and meat products, and antibacterial effects of oregano oil nanoemulsion

Background and Aim: Antimicrobial-resistant Streptococcus species causes several diseases in humans with infection recurrence in some cases. Handling of food under unhygienic conditions resulted in transmission of pathogens to food leading to risks for consumers’ health. This work studies the possible role of humans in transmission of human pathogens to food products. In this study, we focused on the presence of antimicrobial resistance Streptococcus pyogenes, Streptococcus mitis, and Streptococcus pneumoniae found in respiratory patients and in some meat products. We detected the risk factors for patient’s infection and examined the antibacterial effect of oregano oil nanoemulsion (ONE) at different concentrations against the multi-drug resistance strains of streptococci. Materials and Methods: Microbiological culture and polymerase chain reaction were used to identify S. pyogenes, S. mitis, and S. pneumoniae in 120 throat swab samples and 200 meat products. The disk diffusion method was used for investigating the prevalence of resistance against 10 antimicrobials that are commonly used in the treatment of humans and animals. ONE at four concentrations (0.5%, 1.0%, 2.0%, and 3.0%) with an average particle size of 45.4 nm was identified using transmission electron microscopy. The effect of ONE on Streptococcus species isolates was examined using the well diffusion method. Risk factors for human infection were detected using statistical analysis. Results: S. pyogenes, S. mitis, and S. pneumoniae were detected in 14 (11.7%), 9 (7.5%), and 7 (5.8%), respectively, of 120 throat swabs of patients. These bacteria were also detected in 11 (5.5%), 8 (4%), and 6 (3%), respectively, of 200 meat products samples. Beef  luncheon had the highest infection rate for the three species of streptococci. The majority of these isolates showed resistance to antimicrobials such as tetracycline and amoxicillin/clavulanic acid. ONE had an inhibitory effect on the growth of S. pyogenes, S. mitis, and S. pneumoniae, and its effect was significantly increased at a high concentration (3%), with a mean inhibition zone of 36.45±0.302 mm. Very young and old patients, those with chronic diseases, and those exposed to the infection several times were most susceptible to infection. Conclusion: ONE may be used as a therapeutic agent at safe and specific doses, and also in the food industry, to prevent the spoilage of food and protect human health.


Introduction
Streptococcus species originate from several natural sources, including humans and many animal species, in which they naturally colonize the nasal passage, pharynx, and mucosal surfaces of the mouth and intestinal tract. Streptococcus pyogenes colonizes the skin and throat of humans, and causes several diseases, ranging from mild infections such as tonsillitis, pharyngitis, and impetigo, to severe invasive infections such as septicemia, necrotizing fasciitis, and streptococcal toxic shock syndrome [1]. Streptococcus mitis commensally inhabit the human oral cavity [2]. Although S. mitis is considered to have low pathogenicity, it is associated with serious clinical conditions, such as sepsis, pancreatic diseases, and endocarditis, especially in patients with impaired immunity [3]. Streptococcus pneumoniae colonies the human nasopharynx and oropharynx, and is responsible for several human infections, including otitis media, bronchitis, bacteremia, pneumonia, septicemia, septic arthritis, endocarditis, cellulitis, osteomyelitis, peritonitis, and meningitis [4,5].
The poor hygiene leads to contamination of food during handling and preparation through coughing, sneezing, or from skin lesions, from people infected with human pathogens [6]. Prevention of such contamination is important, particularly in S. pyogenes, since the carriers of this pathogen may be asymptomatic [7].
Streptococci are identified by their morphological characteristics, biochemically , and serologically. Recently, molecular methods for the identification of these species have been developed. The species-specific primer spy1258, used to identify S. pyogenes, is important for the study of the epidemiology, pathogenesis, and treatment of this pathogen [8]. Molecular methods are able to discriminate between S. mitis and S. pneumoniae, which have similar biochemical and immunological characteristics [9]. The sequence of the pheA gene can be used to differentiate S. mitis from other members of the viridans Streptococcal group, and is markedly different from the sequence of the gene in S. pneumoniae [10]. The sequence of the Spn9802 gene of S. pneumoniae is specific to S. pneumoniae and does not exhibit cross-reactivity with other species [11].
The acquisition of antimicrobial resistance by streptococci increases their pathogenicity in humans and animals, and causes disease recurrence [12]. Essential oils can be used as antimicrobial agents, but direct application of such oils is limited, because they have poor solubility in water, and therefore must be present in large amounts to produce an effect. The use of large amounts of essential oils may negatively affect food flavor. Therefore, encapsulation of oils as nanoemulsions should enhance their stability, bioefficacy, and bioavailability [13]. Oregano oil nanoemulsion (ONE) has been used as a green bactericidal agent against pathogens resistant to antimicrobial agents. ONE is also used as an antioxidant, antibiofilm, and anti-inflammatory agent in drug delivery systems and skincare medications. In the food industry, it is used as a food preservative to improve its shelf life [14].
This work illuminates the role of humans in the contamination of food products by human pathogens. In this study, we focused on the presence of three pathogenic streptococci found in patients with upper respiratory infections and in some meat products. We detected their antimicrobial responses and the risk factors for infection. We also explored the role of ONE as a natural antibacterial against drug-resistant strains of streptococci.

Ethical approval and Informed consent
This approval was obtained from Ethics Committee of Sohag University Hospital. Patients' participation and collection of samples were done after their consent or their guardian's consent.

Study period and location
This study was conducted from December 2019 to January 2021 in Sohag city which is located in Sohag Governorate in Upper Egypt. All samples were processed for bacteriological examination in the laboratory of Food Hygiene Department, Faculty of Veterinary Medicine, Sohag University.

Study design
Samples from humans, beef luncheon, basterma, beef burger and beef kofta were examined for the presence of S. pyogenes, S. mitis, and S. pneumoniae using bacteriological examination, followed by polymerase chain reaction (PCR), based on the detection of genes specific for each species. All isolates of the three species identified in human and meat products were examined for their resistance against 10 antimicrobials which are generally used in human and veterinary therapeutics using the disk diffusion method. Then, the isolates were exposed to ONE at different concentrations to study its effect, using the well diffusion method. Data were collected from patients, to identify factors related to their infection.

Collection of samples
One hundred and twenty throat swabs were collected from patients admitted to Sohag University Hospital with upper respiratory tract infections such as tonsillitis and pharyngitis. Two hundred samples of meat products, including 50 samples of each of ready-to-eat beef luncheon and basterma, and frozen beef burger and beef kofta were purchased from different markets and groceries in Sohag city.All samples were transported directly to the laboratory for bacteriological examination. A standard form was used for collection of patient data with respect to age, gender, medical, and infection history.

Microbiological examination of samples
Meat samples were prepared as previously described [15]. Human and meat samples were incubated in azide dextrose broth (HiMedia, India) for 24 h at 37°C. Then, samples were cultured on selective Streptococcus agar (Becton Dickinson) containing sheep blood. The colonies were examined by Gram staining and biochemical tests such as catalase, urease, haemolysis, motility, and sugar fermentation like arabinose, fructose, glucose, lactose and mannitol [16].

Molecular identification of Streptococcus species
DNA was extracted from the suspected pathogenic isolates of human and meat products using QIAamp DNA Mini Kits (Qiagen, Hilden, Germany). PCR was used for the detection of the spy1258 gene specific for S. pyogenes, using the primer sequences F: AAAGACCGCCTTAACCACCT and R: TGGCAAGGTAAACTTCTAAAGCA with 407 bp [17], the pheA gene specific for S. mitis, with the primer sequences F: TGGCTTATC CTTCCTAGATGG and R: GATTGCGGTCGACAA A with 557 bp [10], and the spn9802 gene, specific for S. pneumoniae, with the primer sequence F: CAA GTC GTT CCA AGG TAA CAA GTC T and R: CTA AAC CAA CTC GAC CAC CTC TTT (Metabion, Germany) with 162 bp [18], using a thermal cycler (Bio-Rad, Hercules, CA) from Applied Biosystems (Foster City, CA). Some modifications in the cycling conditions of the PCR were made, as follows: For the spy1258 gene, 2 min at 94°C, followed by 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and then a final extension for 5 min at 72°C. The pheA gene regime was 5 min at 94°C, 30 cycles at 94°C for 30 s,58°C for 1 min, and 72°C for 45 s, and then 10 min at 72°C. The spn9802 gene regime was 2 min at Available at www.onehealthjournal.org/Vol.7/No.1/17.pdf 94°C, followed by 25 cycles at 94°C for 15 s, 57°C for 15 s, 72°C for1 min, and a final extension for 5 min at 72°C. The products were examined using 1% agarose gel electrophoresis with ethidium bromide and visualized using a light transilluminator from Biometra (Analytik Jena Group, Jena, Germany).

Antimicrobial sensitivity tests
The disk diffusion method was performed using Müeller-Hinton agar using antimicrobial discs from Oxid Corporation, UK, according to Quinn et al. [16]. The strains of streptococci isolated were tested against 10 antimicrobials from several groups. The zones of inhibition were measured, and the results interpreted based on Clinical and Laboratory Standards Institute [19] guidelines.

Preparation and characterization of ONE
Oregano oil and Tween 80 were mixed at a 2:1 ratio, dissolved in deionized water, and mixed using a magnetic stirrer (Dai Han, Seoul, Korea) for 10 min. The resulting emulsion was sonicated using an ultrasonic homogenizer [20]. Nanoemulsion was prepared in the Nanotechnology Research Unit, Animal Health Research Institute, Cairo, Egypt. The size of the nanoemulsion particles was measured using transmission electron microscopy in the Electron Microscopy Unit, Assiut University.

Effects of ONE
Streptococcus isolates were cultured in selective Streptococcus agar medium for 24 h at 37°C, then 1×10 6 CFU/mL were streaked on Müeller-Hinton agar containing nanoemulsion at concentrations of 0.5%, 1.0%, 2.0%, and 3.0%, using the well diffusion method with 50 µl in each well. The effect of the nanoemulsion was measured by the growth of the inhibition zone [21].

Statistical analysis
The effect of ONE was reported as mean and standard error, calculated using SPSS 14. Pearson correlation was used for detecting the risk factors for infection. The threshold for identification of significant differences was set to p<0.05.

Results
Among 120 patients with a respiratory infection, S. pyogenes was present in 14 (11.7%), S. mitis in 9 (7.5%), and S. pneumoniae in 7 (5.8%) ( Table-1). Factors associated with infection were increased in elderly and young patients, those with chronic diseases, and subjects infected more than 1 time (Table-2). S. pyogenes had the highest prevalence (5.5%) in the meat products examined, followed by S. mitis (4%) and S. pneumoniae (3%). Beef luncheon harbored the highest infection rate of all the examined streptococci, in which S. pyogenes  was present in 12%, S. mitis in 8%, and S. pneumoniae in 6% of samples. All three streptococci were detected in all meat products, except for S. pneumoniae which could not be detected in beef kofta (Table-3). The majority of the strains of streptococci isolated from both human and meat products were resistant to more than 1 antimicrobial agent, such as tetracycline and amoxicillin/clavulanic acid, which had the highest prevalence of resistance (Table-4). ONE at concentrations of 0.5%, 1%, 2%, and 3% had an inhibitory effect on the growth of Streptococcus isolates, with mean inhibition zones of 14.84±0.309, 30.07±0.425, 31.29±0.450, and 36.45±0.302 mm, respectively. The inhibitory effect of ONE increased significantly with concentration (Table-5).
Young and elderly patients were found to be highly susceptible to infection. S. pyogenes and S. pneumoniae were also reported in most patients with chronic disease, especially diabetes mellitus (Table-2). The majority of patients had been infected several times, especially in the case of S. pyogenes, indicating the presence of antimicrobial resistance. Although tonsillitis and pharyngitis are caused by different microorganisms, the presence of resistant strains of streptococci among patients is an important indication that they are the causative agent.
Using specific primers for discrimination between Streptococcus species is important for   Beef luncheon  50  6  12  4  8  3  6  13  26  Basterma  50  2  4  2  4  2  4  6  12  Beef burger  50  1  2  1  2  1  2  3  6  Beef kofta  50  2  4  1  2  0  0  3  6  Total  200  11  5.5  8  4  6  3  25 12.5  proper diagnosis and effective treatment. As discussed by Dunne et al. [22], spy1258 is a target gene for S. pyogenes only and has not been reported in other streptococci or any other bacteria. S. mitis and S. pneumoniae are closely related immunologically and biochemically, and misidentification can occur during routine microbiological examination. Therefore, specific genetic markers are needed to accurately differentiate between them: The pheA gene is used for S. mitis and the spn9802 gene for S. pneumoniae [10,18]. Respiratory droplets released during sneezing or coughing, and inoculation from the hands, are considered to be the most common routes of dissemination of Streptococcal pharyngitis. Therefore, the presence of human pathogens in meat products is an important indicator of contamination of food by food handlers, which may occur at any step during food processing and handling [28].
The results shown in Table-3 indicate that beef luncheon had the highest microbial load of 26%. Basterma samples harbored the three species of streptococci at a percentage of 12% (4% for each species). The lowest load was reported in beef burger and beef kofta, with a percentage of 6% for each, and S. pneumoniae could not be detected in beef kofta. Increased infection in luncheon and basterma may be related to the mishandling, because we observed that food handlers in food premises working without masks or gloves. Luncheon packages were exposed to food handlers several times during the day, which makes them susceptible to contamination, because this product is sold as slices cut on request. The meat is removed from refrigeration, sliced with a cutting machine, and then returned to the refrigerator. Nasal discharges from food handlers may contaminate surrounding environments and surfaces. Decreased contamination in beef burger and beef kofta may be attributed to the effects of freezing, because keeping products at −20°C kills microorganisms [29].
The majority of S. pyogenes, S. mitis, and S. pneumoniae isolates from human and meat products exhibited resistance to various antimicrobials, especially tetracycline, ampicillin, and amoxicillin/ clavulanic acid (Table-4, Figure-4). These results indicated that these antimicrobials were widely used, in a haphazard manner, as a therapeutic medication, which is an important factor for the development of bacterial resistance, and prolongs the duration of recovery. The presence of resistant isolates in food products enables transmission of this resistance to consumers [30].    Therefore, new and natural antibacterial agents are needed to combat antimicrobial resistance in microorganisms. Such agents may include oregano essential oils, in which bacterial resistance was not observed to occur. ONE causes disturbances in membrane protein and lipid production, and defects in the intracellular osmotic pressure, bacterial cell wall and membrane damage, ATPase activity, and RNA synthesis [14]. Owing to the hydrophobic nature of essential oils, formulation of these oils as nanoemulsions will promotes their effects in food, because nanoemulsions are stable compounds, with large surface area and enhanced release inside the bacterial cell [31].
Some factors interfere with the inhibitory effect of essential oils in food, including the interaction between food ingredients and the essential oil, the type of food, the concentration of essential oil used, the origin of the essential oil (type of plant), and the method used for oil extraction [32]. In this context, nanoemulsions with a particle size of 20-200 nm, have thermodynamic stability, coalescence, flocculation, and increased its distribution in food without adverse interactions with food components [33].
The results of experiments using ONE against Streptococcus isolates from human and meat products are shown in Table-5 and Figure-5. ONE with an average particle size of 45.4 nm (Figure-6) at concentrations of 0.5%, 1.0%, 2.0%, and 3.0% produced an inhibitory effect on the strains isolated, and increased concentrations had significantly increased effects (p<0.05). These results indicated that ONE can be used against human pathogens and food contaminants, and may be favored by the public, due to its natural origin. It also has mucolytic, spasmolytic, cough smoothing, and anti-acne effects [34].

Conclusion
Effective diagnosis of S. pyogenes, S. mitis, and S. pneumoniae using specific primers and detection of their antimicrobial activity is necessary for disease treatment. Prevention of the transmission of these pathogens to food products through food handlers can be enhanced by health education and strict supervision, to control these zoonotic pathogens. The survival of these pathogens in food products should be further studied to identify the effective time and temperature of storage. ONE has an inhibitory effect on Streptococcal species isolated from human and food products. It can, therefore, be used as a human therapeutic and food preservative, but additional research is needed to identify the safe and effective dose.

Authors' Contributions
AAH and NMA equally contributed in the idea, study design, collection of samples and data, literature search, laboratory work, data analysis, and manuscript writing. Both authors read and approved the final manuscript.