Phenotypic Resistance of Staphylococcus aureus to Antibiotics in Dogs of Tamale Metropolis, Ghana

anus, inner ear, and outer ear, were examined. Isolation and antibiotic resistance of S. aureus were determined using the USA Bacteriological Analytical Manual and the Disc Diffusion method, respectively. Results: The presence of S. aureus in the dogs ranged from 8.3% (anus) to 58.3% (nose), averaging 40%. The microbial load also ranged from 2.9 log cfu/cm 2 (mouth) to 3.4 log cfu/cm 2 (outer ear) with an average of 3.2 log cfu/cm 2 . There were significant differences among the examined samples regarding the presence of S. aureus , but not the microbial load. The overall resistance, intermediate resistance, and susceptibility of S. aureus were 46.2%, 12.9%, and 42.2%, respectively. The S. aureus was highly resistant to teicoplanin (88.0%) and susceptible to chloramphenicol (72.0%). The multiple antibiotic indexes ranged from 0 to 0.9, and 89.1% of the isolates exhibited multidrug resistance. Conclusion: The findings of the current study revealed that healthy dogs in Tamale Metropolis, Ghana, were carriers of S. aureus as well as other bacteria, and S. aureus exhibited different resistance patterns to antibiotics.


Introduction
Staphylococcus aureus (S. aureus) is a Gram-positive cocci-shaped bacterium that tends to be arranged in clusters and is a member of the family Micrococcacae 1,2 . It is an important bacterium due to its capacity to induce a wide range of diseases and its ability to adapt to different environmental conditions 2 . The emergence of multidrugresistant strains of S. aureus has contributed to the importance of this pathogen. Staphylococcus aureus could be acquired from human and animal hosts. Approximately 30% of humans bear S. aureus in their nasal cavities, which is the primary reservoir and the primary source of infection 3 . Strains of S. aureus have been reported to cause mastitis in cattle, botryomycosis in horses, dermatitis in dogs, septicemia, and arthritis in poultry 4,5,6,7 . Case reports of human infection or colonization from household pets have shown the high likelihood of animals acting as reservoirs for transmission of this pathogen 8,9,10 .
Pets can develop a social and emotional relationship with humans and their environment 11 . This has led to a strong attachment between dogs and their owners or caretakers, and therefore, pets are treated as family members 11 . Dogs have been reported to be potential sources of various zoonotic pathogens, such as S. aureus, Escherichia coli, Salmonella enterica, Klebsiella species, and Bacillus species 9,12,13,14 that are resistant to various antibiotics, including ampicillin, cephalosporin, gentamicin, enrofloxacin, methicillin, and tetracycline 10,15,16,17 .
In Ghana, dogs are kept for different purposes, such as security, hunting, pets, and even food (meat). Although keeping dogs in Ghana is now rampant and currently raised due to the aforementioned reasons, the closeness between dogs and humans makes them potential sources for the transmission of zoonotic pathogens, such as S. aureus, to humans. However, information on the occurrence of S. aureus in dogs and their antibiotic resistance patterns in Ghana is limited. As these animals are in close contact with humans, the presence of antibiotic-resistant S. aureus can pose a health threat to humans. Therefore, the present study was conducted to determine the presence and antibiotic resistance of S. aureus in apparently healthy dogs in the Tamale Metropolis, Ghana. Furthermore, the microbial load of the various parts of the dogs was determined. To the best of the researchers' knowledge, the current study is one of the first reports on the prevalence of antibiotic resistance S. aureus in dogs of Ghana.

Ethical approval
The present study did not cause any harm to humans or animals and was approved by Project Supervisors of the Department of Veterinary Science, University for Development Studies, Ghana.

Study area
The present study was carried out at the Tamale Metropolis of Ghana. Geographically, the Tamale Metropolis lies between latitudes 9 o 16 and 9 o 34 North and longitudes 0 o 36 and 0 o 57 South 18 . The Metropolis has an estimated total land area of 646.90180 sq km, and a population of 223252 18 .

Sample collection
A total of 120 samples were randomly collected from dogs without any clinical manifestation of diseases in the Tamale Metropolis, confirmed by a veterinarian. Houses that owned dogs were numbered, and the researchers randomly picked the numbers from a box. Houses picked were visited, and with the help of dog owners, such as the restrainers and a veterinary officer, swabs were taken from the various parts of the dogs. The dogs were randomly selected without emphasis on selecting a particular breed. Sterile cotton swabs were used to swab the anus, (n=24), mouth (n=24), nose (n=24), inner ear, (n=24), and outer ear (n=24) of the dogs at their homes with the help of dog owners and a veterinary officer. The swabbed samples were placed in an ice chest box containing ice blocks and transported to the Spanish Laboratory of the University for Development Studies, Nyankpala Campus, Ghana, where they were analyzed for S. aureus. Sampling was carried out from December 2019 to March 2020.

Enumeration of aerobic plate count
Enumeration of aerobic plate count was performed using a slightly modified procedure as previously done 19,20 . Swabs were soaked in 9 ml of 1% buffered peptone water (BPW), and serial dilutions from 10 -1 to 10 -5 were made using one ml of 1% BPW. It was then spread plated (0.1 ml) unto plate count agar and incubated at 37°C for 24 hours. Afterwards, the colonies were counted and expressed in colony-forming units. All media used were purchased from Oxoid, Basingstoke, the UK.

Isolation and identification of Staphylococcus aureus
Isolation and identification of S. aureus were performed using a slightly modified procedure 21,22 . Briefly, swabbed samples from healthy dogs were pre-enriched in BPW and incubated at 37°C for 24 hours. They were then streaked with mannitol salt agar (MSA) and incubated at 37°C for 24 hours. Presumptive S. aureus colonies formed yellow colonies on MSA surrounded by a yellow area. Two or three colonies were picked and purified on Trypticase soy agar and incubated for 24 hours at 37°C. Gram staining and Staphylase test were used to confirm the purified S. aureus colonies. All media and reagents used were purchased from Oxoid, Basingstoke, the UK.

Antibiotics susceptibility of Staphylococcus aureus
The disk diffusion method was used to determine the antimicrobial resistance of S. aureus against some antibiotics, including Amoxycillin/clavulanic acid (Amc, 30 μg), chloramphenicol (C, 30 μg), gentamicin (Cn, 10 μg), ceftriaxone (Cro, 30 μg), ciprofloxacin (Cip, 5 μg), azithromycin (Azm, 15 μg), sulphamethoxazole/ trimethoprim (Sxt, 22 μg), tetracycline (Te, 30 μg), and teicoplanin (Tec, 30 μg) 23 . Pure cultures of S. aureus were grown overnight in tryptic soy broth at 37°C, and the concentration was adjusted to 0.5 McFarland turbidity. About 0.5 ml of the culture was spread plated on Mueller Hinton agar. Four and five antimicrobial disks were placed on the surface of the agar plate at a distance to avoid overlapping inhibition zones. The plates were incubated at 37°C for 24 hours, and the results were interpreted according to a previous study 24 . All media and disks used were purchased from Oxoid, Basingstoke, the UK. The multiple antibiotic resistance (MAR) index was calculated and interpreted to use the formula a/b, where a represents the number of antibiotics to which a particular isolate was resistant, and b denotes the total number of antibiotics tested 25 .

Data analysis
The data obtained from microbial load was analyzed using ANOVA of GenStat Software 12.1 Edition. Data on the presence of S. aureus were analyzed using binary logistic of IBM Statistical Package for the Social Sciences (SPSS) Software Version 17. The statistical difference test was done using the Wald test in Chi-square. All differences were determined at a 5% significance level. Table 1 shows the microbial load of the anus, mouth, nose, inner ear, and outer ear of apparently healthy dogs in the Tamale Metropolis. The microbial load was 3.2 log cfu/cm 2 (anus), 2.9 log cfu/cm 2 (mouth), 3.2 log cfu/cm 2 (nose), 3.1 log cfu/cm 2 (inner ear), and 3.4 log cfu/cm 2 (outer ear). There were no significant differences in microbial load among the anus, mouth, nose, inner ear, and outer ear (p > 0.05).

Microbial loads of healthy dogs in the Tamale Metropolis, Ghana
In Ghana, the increase in the number of dog owners has caused close contact between humans and dogs. Such close contact enhances the transmission of pathogens. The current study showed that bacteria were present in the outer ear, anus, nose, inner ear, and mouth of the dogs studied. These bacteria could be naturally present in the parts of the dogs examined, or the dogs might pick them from their food, environment, and humans. In support of this, various types of bacteria in dog feces collected from urban streets, could be considered a risk factor for the transmission of microorganisms among humans, the environment, and dogs 26 . In another study, diverse bacterial species in the mouth of dogs were observed 27 . Dog foods have also been reported as potential sources of bacteria that can cross-contaminate the mouth and other parts of the body 28 .

The presence of Staphylococcus aureus in healthy dogs of the Tamale Metropolis, Ghana
The presence of S. aureus in the apparently healthy dogs in the Tamale Metropolis is presented in Table 2. The overall presence of S. aureus in the dogs was 40% (48/120). Staphylococcus aureus was most common in the nose (58.3%), followed by the mouth (54.2%), outer ear (50.0%), inner ear, (29.2%), and anus (8.3%). Significant differences were observed in the presence of S. aureus in the dog samples (p < 0.05). Mouth, nose, and outer ear did not differ significantly (p > 0.05) from each other but were significantly higher (p < 0.05) than the anus. The inner ear did not differ significantly (p > 0.05) from the anus, mouth, and outer ear except the nose. Similar to the microbial load, S. aureus was also present in all the various parts of the healthy dogs examined. The contamination of S. aureus was generally higher in the dogs' mouth, nose, and outer ear. The sources of S. aureus include the environment (air, soil, water) as well as skin and nose 1,9 . This pathogen can be transferred from these sources by direct or indirect means. For instance, it is common to find dogs licking themselves or their owners, potentially transferring S. aureus to other body parts. The licking of dog owners also serves as a potential means of transmitting S. aureus between humans and dogs and vice versa. Another study supports this by indicating few dog-to-dog and dog-tohuman transmissions of S. aureus 17 15 Differences in the location of examined organs, and handling methods of dogs accounted for the differences in the prevalence rates.
Dogs can put humans at health risk because they can be carriers of zoonotic antibiotic-resistant bacteria 33 . In the current study, S. aureus was resistant to teicoplanin, ceftriaxone, azithromycin, and tetracycline (> 50%). They were susceptible to chloramphenicol, sulphamethoxazole/ trimethoprim, ciprofloxacin, and gentamicin (≥ 60%). This suggests that teicoplanin and tetracycline will not be the antibiotic of choice for treating S. aureus infections associated with dogs in the Tamale Metropolis. However, chloramphenicol could be used to manage infections in dogs caused by S. aureus. It was observed that S. aureus obtained from dogs was resistant to gentamicin (40.2%), tetracycline (75%), and ciprofloxacin (7.7%) 12 . The present study found lower resistances to gentamicin and tetracycline except for ciprofloxacin. Moreover, coagulase-positive Staphylococci from 112 dogs sampled exhibited 23.2%, 21.4%, 9.8%, and 2.7% resistances to tetracycline, trimethoprim/sulfamethoxazole, ciprofloxacin, and chloramphenicol, respectively, which were higher in the current study 10 . High resistance to tetracycline (87.5%) and chloramphenicol (75.0%) for S. aureus isolated from dogs were recorded in another study, compared to the current study 34 . Differences in the extent to which antibiotics were used and handled in the study areas contributed to differences in the results obtained.

Antibiotic resistance profile and multiples antibiotic resistance index of individual Staphylococcus aureus recovered from healthy dogs in the Tamale Metropolis, Ghana
The antibiotic resistance profile and MAR index of S. aureus from the healthy dogs are shown in Table 4. Two, four, and three S. aureus isolates were resistant to eight, seven, and five different antibiotics, respectively. Resistance to three or more different antibiotics (multidrug resistance) was recorded for 90 isolates (89.1%). Multidrug resistance S. aureus isolates have also been reported in dogs 5,12 . Multiple antibiotic resistance ranged from 0 (resistant to 0 antibiotics) to 0.9 (resistant to 8 antibiotics). Staphylococcus aureus with a MAR index of greater than 0.2 originates from sources where antibiotics are frequently used, while those with MAR less than 0.2 originates from sources where antibiotics use is uncommon 35 . Based on this, 72% of the isolates originated  from sources where they were frequently exposed to antibiotics. The S. aureus isolates exhibited 19 different phenotypic antibiotic-resistant profiles. The resistant profile Tec-Cro was the most common and was exhibited by three different S. aureus isolates. Staphylococcus aureus of the anus (8A) and month (9M) shared the same phenotypic resistance pattern (Amc-Azm-Tec-Te). Similarly, S. aureus with codes 24N and 23M shared the same phenotypic resistance pattern (Amc-Tec-Cro). These suggest possible cross-contamination; nonetheless, molecular characterization is required to authenticate this observation.

Conclusion
Bacteria were present in healthy dogs' anus, mouth, nose, inner ear, and outer ear. Furthermore, S. aureus was recovered from these parts. The S. aureus exhibited some resistance and intermediate resistance to some antibiotics. Therefore, healthy dogs in the Tamale Metropolis, Ghana, are potential sources for the transmission of S. aureus resistant to some antibiotics.