Bacteriological and Molecular Comparative Study between Staphylococcus aureus Isolated from Animals and Human

Staphylococcus aureus represents a serious health hazard on both animals and humans. The main goal of the present work was to compare between S. aureus isolated from animal and human origin. This was achieved through studying the virulence, phenotypic characterization, genotypic testing of mecA gene, analysis the antibiotic resistance profile of the recovered S. aureus isolates. A total number of 165 samples were collected from clinical mastitic cows and sheep, pus from abscesses collected from septic wounds of infected animals, respectively. Moreover, blood, pus swabs from abscesses and septic wounds, and sputum samples were collected from diseased humans from Assiut Governorate, Egypt. The results revealed that incidence of S. aureus isolates recovered from the examined animal samples were 8.33%, 100% and 20%, per, while from human cases (blood, pus and sputum) were 16.67%, 53.49% and 75%, independently. Using Staphaurex kits, the prevalence of coagulase positive S. aureus from animal samples reached 8.33%, 100% and 20%, but from the diseased human cases were 16.67%, 53.49% and 75%, respectively. Antimicrobial Sensitivity Testing of the animal isolates were resistant to cefoxitin (15.79%), tetracycline (10.53%), clindamycin and erythromycin (10.53%), while the isolates were sensitive to gentamicin (100%), trimethoprim + sulfamethoxazole and vancomycin (94.74% for each). However, S. aureus human isolates were resistant to cefoxitin (75%) and tetracycline (78.57%) and sensitive to vancomycin (100%), ciprofloxacin (89.29%) and trimethoprim+ sulfamethoxazole (82.14%). Out of 47 identified S. aureus strains, 3 from animal isolates (15.79%) and 21 from human isolates (75%) proved to be methicillin-resistant (MRSA). Furthermore, one animal isolate (5.26%) and 12 human isolates (42.86%) were multi-drug resistant (MDR). S. aureus isolates from animal and humans were subjected to genotypic characterization of mecA gene using PCR. All the animal and human isolates were positive for mecA gene with a percentage of 100%. The results of this study provide that from both animals and human samples; the isolation rate of S. aureus was greater than that of MRSA followed by MDR. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Staphylococcus aureus is a commensal microorganism and the most virulent of all staphylococci (Jenkins et al., 2015). S. aureus strains produce a wide spectrum of protein toxins and virulence factors, which contribute to the pathogenicity of this organism so, pathogenic to both human and animals (Zecconi and Scali, 2013). In bovines, it is responsible for intra-mammary infection and milksafety problems, in dairy herds, it is the main etiological agent of contagious clinical/sub-clinical mastitis (Taverna et al., 2007) because it is very well adapted to colonize on the teat skin and teat canal, as milk is a very suitable medium for the growth of many pathogens (Nam et al., 2011).
In human, it causes range of illnesses such as Staphylococcal Scaled Skin Syndrome, toxic shock syndrome and food poisoning by releasing superantigens into the blood stream (Todar, 2005), pneumonia, phlebitis, mastitis, meningitis and deepseated infections, such as osteomyelitis and endocarditis (Morya et al., 2012), septic arthritis (Edwards and Massey, 2011), in addition to be the major cause of numerous hospital and community acquired infections (Gill et al., 2005).
The development of these secondary infections is due to bacterial dissemination from the blood into the surrounding tissues and is associated with significantly increased morbidity and mortality (Edwards and Massey, 2011).
MRSA characteristic phenotype is due to an extra penicillin-binding protein (PBP2a)  The purpose of the present study was to investigate phenotypic and genotypic characterization of S. aureus isolates recovered from animals and humans. This was achieved by detection of their hemolytic activity, investigating their clumping factor, protein A and capsular polysaccharides using Staphaurex kits, detection of: coagulase enzyme by coagulase test, analysis the antibiotic resistance profile, and genotypic characterization of mecA gene using PCR technique of the recovered S. aureus isolates.

Sampling
All the samples were collected under sterile conditions. A total of 165 samples (84 from animals and 81 from humans). The animal samples were collected from clinical mastitis cows' milk (n=40), clinical mastitis sheep's milk (n=14), and pus from septic wounds and abscesses of different animals (n=30). The human samples were collected from diseased human admitted to clinics of Assiut University Hospital in Assiut Governorate, Egypt (blood samples were collected in blood culture bottles), blood (n=13), pus (n=61) septic wounds, abscesses, and sputum (n=7). Then, samples were cultured on nutrient agar for pigmentation and mannitol salt agar then incubated at 37ºC for 24h. The grown colonies were examined microscopically.

Preparation of the collected samples for isolation and identification of S. aureus
Animal quarter milk samples were identified according to NMC (2004).

Isolation of Staphylococcus spp.
Recovered from the collected samples was done according to Bailey and Scott (1974). The suspected bacterial colonies showing typical colonial appearance were picked up and stabbed into semisolid media and incubated at 37ºC for 24 hours for another investigation.

4.Identification and characterization of S. aureus isolates (Quinn et al., 2002)
The suspected colonies were inserted into semisolid agar tubes for further identification and preservation of the isolates. They were identified according to diverse biochemical tests including pigmentation, Gram -staining, catalase, coagulase and hemolysis.  The DNA amplification was performed as Initial denaturation (94°C for 4 min) then 30 cycles of (denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 72°C for 1min) then final extension at 72°C for 4min. Negative control reactions without any template DNA were carried out.
The procedure of agarose gel electrophoresis for identification and screening of the PCR products was carried out according to Sambrook et al., (1989) using 100 bp DNA ladder (Vivantis-Malaysia).  Results of antimicrobial sensitivity testing of S. aureus recovered from the examined samples All S. aureus isolates recovered from animal samples (19 isolates) as well as those recovered from human samples (28 isolates) were tested for their antimicrobial sensitivity.   Coagulase is one of the diagnostic enzymes produced by S. aureus. It was used to indicate their virulence. However, after performing coagulase and Staphaurex kits, the present study showed that, from the 81 examined diseased human samples, 59 isolates were staphylococci spp. (72.8%). Out of 12 (92.3%) positive staphylococci from human blood, only 2 were S. aureus (16.7%), out of 43 (70.5%) positive staphylococcal pus samples from human septic wound and abscesses, only 23 were S. aureus (53.5%) and out of 4 (57.1%) positive staphylococcal sputum samples, only 3 were positive (75%). So, the total S. aureus isolates from the present work was 28 isolates (47.5%) and the coagulase negative staphylococci (CoNS) were 31 isolates (52.5%) represented as; 20 isolates from the pus samples (46.5%), 10 isolates from the blood samples (83.3%) and 1 isolate from the sputum samples (25%). These findings were higher than those recorded by El-Jakee et al., (2008) who estimated staphylococci from the urine of infected urinary tract, septic wounds and nasal swabs from humans (36%  (2015) reported that the occurrence of S. aureus in clinical mastitis (73.3%) was higher than that in the subclinical mastitis (42%).

RESULTS
In the present study, the obtained CoNS was 66.7%, which was higher than that obtained by Bradley et al., (2007 The present study revealed that the 19 S. aureus isolated from animal samples were examined by the antimicrobial sensitivity test, and revealed high sensitivity against gentamicin (100%). This finding agreed with that of Negash (2015) who reported that all animal isolates were found susceptible to gentamicin (100%). Abo-Zaid and Bahout (1993) estimated that gentamicin was the antibiotic of choice in the treatment of 86.7% of the mastitis cows. Akram et al., (2013) reported that gentamicin was effective in the treatment of bovine mastitis with an incidence of 80.4%.
In addition to Gentamicin, the animal isolates were sensitive to trimethoprim+ sulfamethoxazole and vancomycin (94.7% for each), ciprofloxacin (89.5%) and cefoxitin and clindamycin (84.2%), and was highly resistant to cefoxitin and tetracycline with a percent of 15.8%, and clindamycin and erythromycin 10.5% each, but were intermediate sensitive to tetracycline (31.6%) and erythromycin (21.05%). These results indicated the need to use prophylactic treatment and control measures of mastitis for heifers and cows as confirmed by Castelani et al., (2015). Also, a higher resistance of cefoxitin (42.7%) and tetracycline (77.4%) were reported by Negash (2015).
On the other hand, 28 S. aureus isolates from the diseased human cases were tested for antimicrobial sensitivity, and showed high sensitivity to vancomycin (100%), ciprofloxacin (89.3%) and trimethoprim+ sulfamethoxazole (82.1%), and clindamycin (78.6%), where S. aureus isolates were highly resistant to tetracycline (78.6%). Also, 5 isolates showed intermediate sensitivity was recorded against tetracycline (17.9%) and clindamycin and trimethoprim + sulfamethoxazole with a percentage of 7.1% for each. The obtained results revealed that S. aureus isolated from diseased human was highly resistant to cefoxitin with an incidence of 75%, a higher resistance to cefoxitin (100%) was reported by Davoodabadi et al., The study obtained results reflect the extent of misuse of antibiotics in the treatment of bacterial infection in both animals and humans, which may lead to antibiotic resistance. That agrees with Frieden (2013) who stated that the animal, the environment and the infected persons play a vital role in the spread of the bacteria and also with Apparao et al., (2009) who mentioned that the antibiotic selected for the treatment of mastitis has often been based on results of invitro susceptibility testing.
The present study concluded that 3 out of 19 S. aureus isolates were found to be MRSA strains (15.79%) isolated from animal samples. Higher rates were recorded in diseased human, where 21 out of 28 S. aureus isolates were found to be MRSA strains (75%). So, the total number of MRSA isolates was 24 out of 47 isolated from all the examined samples in the present study. It was such a higher rate than recorded by Nsofor et al., (2016) (38.5%). MRSA was detected among S. aureus hospital-acquired (HA) infections in Italy (41%) as revealed by Monno et al., (2003). On contrary, a very lower rate was detected among secondary school students, where 2.04% of the students were found to be MRSA carriers (Habeeb et al., 2014). MRSA was detected in 32% of the Egyptian outpatients while it was found in 25% of the Saudi Arabian outpatients (Abou Shady et al., 2015), 47% in India and 90% in Latin American hospitals (CDDEP, 2019), 28.6% (genotypicaly MRSA) in Uganda (Abimana et al., 2019).
Regarding the results of MRSA detected in animal samples (15.85%), higher rates (36.6%) were recorded by Tesfaye (2014) and Helal et al., (2015) who recovered MRSA among S. aureus isolated from clinical mastitis cases (36.6%) and sheep abscess and bumble-foot samples (100%) and mastitic milk samples 77.7%, depends on the context itself. MDR S. aureus is a complicated problem and is difficult to be eliminated due to the presence of numerous antibiotics resistance genes (Anitha et al., 2016). The obtained results showed that 1 (5.3%) out of 19 S. aureus strains from the examined animal isolates were found to be multidrug-resistant (MDR) (5.26%). In contrast, higher rates were recorded by Kim et al., (2003) and Negash (2015) from animal cases with a percentage of 51% and 45.3%, respectively. In addition, 12 out of 28 S. aureus strains were found to be MDR strains (42.9%). However, MDR isolation rate was higher in strains recovered from diseased human than that of the examined animals, showing resistance to three or more antimicrobial agents as cited by Intrakamhaeng and Komutarin (2012).
The clinical administration of antibiotics, against the pathogenic bacteria has gradually stopped due to emergence of MDR bacterial strains including S. aureus (Efuntoye et al., 2011).

CONCLUSION
From the aforementioned results, there was a significant increase in S. aureus isolation rate in the examined clinical mastitis sheep's milk compared with examined clinical mastitic cows' milk and pus samples of different animals. S. aureus strains were highly resistant to cefoxitin and tetracycline followed by clindamycin and erythromycin, which were isolated from infected animals. In diseased human, the strains were highly resistant to tetracycline and cefoxitin, resulting in methicillin-resistant S. aureus (MRSA) problem. Also, gentamicin, trimethoprim+ sulfamethoxazole, and vancomycin were the most effective antibiotics that can be used in the treatment of S. aureus infections in field animals, while in diseased human, the most suitable antibiotics for treatment of S. aureus infections were vancomycin, ciprofloxacin and trimethoprim+ sulfamethoxazole. On the other hand, S. aureus is susceptible to vancomycin and ciprofloxacin so, both drugs are the most frequent drug of choice to treat a variety of S. aureus infectious diseases for humans.

Declaration of Competing interest
On behalf of all authors, I hereby declare that no conflict of interest may interfere with the publication of the manuscript.