Investigation of antibiotic resistance pattern of Staphylococcus aureus in clinical samples of animals and humans from selective areas of Bangladesh

Background : Staphylococcus aureus ( S. aureus ) is a potential pathogen responsible for producing various infectious diseases. Determination of the prevalence of S. aureus infections both in animals and humans and elucidation of their antibiotic resistance pattern is crucial. The objective of this study was to find out the prevalence of Staphylococcal diseases in animals and humans, and their resistance pattern to commonly used antibiotics. Methods: A total of 100 animal and 100 human clinical samples were analyzed by traditional method. The S. aureus was identified by their cultural characteristics, gram’s staining, and catalase and coagulase tests. Antibiotic resistance pattern of the isolates was determined by disc diffusion method using various types of antibiotics. Results: The prevalence of S. aureus in animals and humans were 54% and 40%, respectively. S. aureus isolates of animal origin were highly resistant against penicillin (64.81%) and oxytetracycline (42.59%), and the lowest resistance was against oxacillin (7.40 %). S. aureus isolates from human were also showed a higher percentage of resistance against penicillin (87.5%) then oxacillin, cloxacillin, amoxicillin (37.5% each), and lowest resistance was observed against fusidic acid (5%). Conclusion : The findings of this study will certainly help veterinary clinicians or physicians to select appropriate antibiotics like oxacillin and fusidic acid for the treatment of different types of staphylococcal infections.


Introduction
S. aureus is the most common cause of various types of infections known as staphylococcal infections.This organism was found commonly as a commensal on the human skin such as on the scalp, armpits, groins, nose (~25% humans), throat and may also be found less commonly in the colon and urine (Rahman et al., 2005;Islam et al., 2007;Gurung et al., 2020).They can also be found as commensals on domestic animals such as cattle, horses, dogs, cats, and other animals.Sir Alexander Ogston, a renowned surgeon, discovered S. aureus in the pus from surgical abscesses in Aberdeen, Scotland in 1880.S. aureus is a Gram-positive coccus which produces grape-like clusters under the microscope.On blood agar plate, the organism looks like round, largened, golden-yellow colonies with characteristic hemolysis (β-types).
The organism is responsible for causing both minor infections to major infections (lifethreatening) in both humans and animals.The minor infections include pimples, impetigo, furuncles/boils, folliculitis, carbuncles (a collection of furuncles), cellulitis, and abscesses (Nwachukwu et al., 2009;Gurung et al., 2020).The life-threatening diseases include endocarditis, meningitis, pneumonia, toxic shock syndrome, septicemia, and septic arthritis, osteomyelitis etc. (Islam et al., 2019;Islam et al., 2020), and in chickens, it causes bumble foot.S. aureus secretes several toxins, depending on the strain.The organism causes a severe disease named staphylococcal scalded skin syndrome in infants.S. aureus is known as one of the crucial causal agents of mastitis in dairy animals.
The antibiotic resistant S. aureus has been unknown when penicillin first introduced in 1943 for the treatment of infectious diseases.(Islam et al., 2008;Chambers and DeLeo, 2009).Shortly after introducing antibiotics (e.g., by 1950) about 40% hospital isolates of S. aureus were resistant against penicillin and this had risen to 80% by 1960 (Rahman et al., 2005).At present, S. aureus has been found resistant to many commonly used antibiotics.Nowadays, due to the penicillinase (a form of β-lactamase), a very low percentage of S. aureus isolates were sensitive to penicillin globally (Islam et al., 2011).Then methicillin, flucloxacillin, oxacillin, and other β-lactamaseresistant penicillin were developed for the treatment of penicillin-resistant S. aureus which was used as first-line antibiotic agents (Ahmed et al., 2019).At present, there have been reports of resistance of S. aureus against βlactamaseresistant penicillin (Chambers and DeLeo, 2009).
Resistance against antibiotic agents in bacteria was developed by bacterial gene mutations, gene rearrangement, or by the acquisition of resistant genes from other antibiotic-resistant bacteria by horizontal transfer (Dutta et al., 2013;Foster, 2017).Penicillin resistant S. aureus were developed by the production of penicillinase, the enzyme which breakdowns the β-lactam ring of the penicillin antibiotic (Lowy, 2003;Islam et al., 2011).Methicillin-resistant bacteria were developed by the acquisition of the mecA gene (Chambers and DeLeo, 2009;Dutta et al., 2013).The mecA gene encodes for an alternate penicillin-binding protein (PBP-2') which has a lower binding to β-lactam antibiotics (Islam et al., 2011).Thus, methicillin resistance in bacteria also develops resistance to all other β-lactam antibiotics (Ayukekbong et al., 2017).If the bacteria acquire the vanA gene then it becomes resistant against glycopeptides.The vanA gene codes for an enzyme which is responsible for producing alternative peptidoglycan to which vancomycin is not able to bind and ultimately treatment failure occurs (Dutta et al., 2013;Foster, 2017).
Infectious diseases caused by S. aureus and the susceptibility of S. aureus to commonly used antibiotics are a major concern worldwide among human and veterinary medicine for effective therapy.But a scarcity of surveillance and accuracy of the source of samples, methods of bacterial identification, methods of antibiotic susceptibility testing, guidelines for the interpretation of antibiotic susceptibility etc. of different studies have existed in many countries of the world (WHO, 2014; Chambers and DeLeo, 2009).The present study was undertaken to find out the prevalence of staphylococcal diseases in animals and humans, and their resistance pattern to commonly used antibiotics.The findings of this study are crucial to search for antibiotics effective for clinical S. aureus treatment.

Sample collection and preparation
Samples comprising wound swabs, pus, and various exudates were collected aseptically from different animals and humans.After cleaning the wounds with normal saline and removing the debris, swabs have taken using sterile swab sticks by applying moderate rubbing.Pus samples were also collected by immersing the swab sticks into the pus.From each patient a single type and a single sample was taken.After collection, the swabs, the swab sticks were put into sterile test tubes containing 10 ml nutrient broth medium and shifted to the laboratory for incubation.
The liquid samples such as the mastitic milk was also collected by strict aseptic precautions from the field.The teats were cleaned by normal saline just prior to collection of samples.Then milk was kept into a screw-capped vial and kept in an ice flask immediately.The samples were then kept in 4ºC temperature for few days if required.The samples from the refrigerator were carried to the laboratory by using ice flask.In the laboratory, the samples were inoculated into the Nutrient broth media.The animal origin clinical samples were collected from Bangladesh Agricultural University, Mymensingh and Milk Vita, Sirajganj, Bangladesh and human clinical samples were collected from Mymensingh Medical College Hospital (MMCH), Mymensingh, Bangladesh from January to June 2007.

Isolation and identification of S. aureus Isolation and identification
Nutrient broth media of various types of clinical specimens were incubated in an incubator.When bacteria were grown, there was found the development of turbidity and sometimes formation of sediment after 24 hours.The positive clinical samples then subsequently recultured into the Nutrient agar and Blood agar media.On Nutrient agar media, 2-4 mm circular, golden yellow pigment producing, and convex shining surface colonies were detected as S. aureus.On Blood agar media, yellow to cream or sometimes white colonies with 1-2 mm in diameter showing zone of β hemolysis after overnight incubation was suspected as S. aureus.All the media were incubated for 24 hours at 37ºC in the incubator.

Gram's staining method
Gram's staining method was applied as described previously (Tenover and Hirschmann, 1990).After staining, the slide was examined under a microscope.

Subculturing on Mannitol Salt agar media
On Mannitol Salt Agar media, S. aureus was cultured and bacteria produce large colonies surrounded by yellow zones which are characteristic for S. aureus.

Biochemical tests
The biochemical test was applied as described previously (Cheesbrough, 2000).Both catalase and coagulase tests were used for confirmation of S. aureus.

Antibiogram study
Antibiogram study for the isolated S. aureus was performed by disc diffusion method (Kirby-Bauer technique) (Bauer et al., 1966) as per the recommendation of National Committee for Clinical Laboratory Standards (NCCLS, 1997).Various types of antibiotics were tested, and the tests were performed on Mueller-Hinton agar plate.In brief, suspension of S. aureus was prepared to have equivalent turbidity to 0.5 McFarland standards.Then the suspension of S. aureus was taken out by a cotton swab squeezing on the wall of the test tube.Then the surface of the Mueller-Hinton agar plate was lightly and uniformly inoculated by cotton swab (Islam et al., 2008).The inoculated plates were incubated at 35ºC for 24 hours.After incubation, the zone of inhibition of specific antimicrobials on agar plates was measured (NCCLS, 1997).Then the results were interpreted as sensitive (S) or resistant (R) according to the reference zone of inhibition of specific antibiotics.The known strain e.g., ATCC 25923 of S. aureus has used as a control for maintaining the quality.

Results
The cultural characteristics, staining characteristics, and biochemical characteristics of S. aureus shown in Fig. 1.The antibiotic susceptibility pattern of isolated S. aureus shown in Fig. 2. A total of 100 animal and 100 human clinical samples were examined.Majority of the animal's origin specimens were collected from cattle 87 (87%) followed by goats 10 (10%) and then buffalo 3 (3%).Most of the specimens were mastitic milk 62 (62%) followed by pus 21 (21%), wound swab 17 (17%) (Table 1).All the culture-positive specimens were catalase positive.
The overall prevalence of S. aureus in animal samples was 54%.However, the prevalence of S. aureus in mastitic milk, pus and wound swab were 39%, 8% and 7% respectively (Table 1).The human clinical samples were collected from hospital admitted patients.The distribution of human samples was presented in Table 2.All the culture-positive specimens were catalase positive.The overall prevalence of S. aureus in human specimens was 40%.However, the prevalence of S. aureus in different human specimens varied from 20-100% (Table 2).
Antibiotic resistance pattern of animal origin S. aureus isolates was presented in Table 3 (n = 54).
The highest resistance was found against penicillin (64.81%).Antibiotic resistance pattern of human origin S. aureus was shown in Table 4.Most isolates of human samples were resistant to penicillin (87.5%).

Discussion
The prevalence of S. aureus in human and animal samples were determined in this study.Moreover, the antibiogram of the isolates were also studied.Oxacillin and fusidic acid for the treatment of animal and human diseases due to S. aureus can be recommended based on their antibiogram profile.The diseases caused by S. aureus in the animal population are quite high (54%).A similar finding is also reported in a recent study (Anueyiagu et al., 2020).The most likely reason for the higher prevalence might be due to the commensal nature of S. aureus.They can produce fatal infections to animals during stress conditions (Rahman et al., 2005;Chambers and DeLeo, 2009;Islam et al., 2011).Among all the animal specimens, the prevalence of staphylococcal mastitis was 62.90%.Several authors also reported similar prevalence of staphylococcal mastitis (Plozza et al., 2011;Abebe et al., 2016).The higher prevalence of staphylococcal mastitis in the animal population may be due to substandard hygiene, high density of milking cows, lack and/or no teat dipping after milking (Abebe et al., 2016).It has been revealed that milk from the cows affected by mastitis have a potential public health significance.So, measures should be taken to prevent mastitis caused by S. aureus.
Half of the human clinical specimens were wound swabs which indicates that wound is a common affection among clinical patients admitted in the hospital.In favorable conditions, aureus might produce abscess on the wound and ultimately there might be septicemia and other fatal infections (Rahman et al., 2005;Nwachukwu et al., 2009).Previously, swabs from wound were also reported as major specimens in the bacteriological laboratory by other authors (Pandey et al., 2012;Gurung et al., 2020).The higher proportion of the positive culture of wound specimen might be due to the higher number of wound infection in clinical patients in hospitals.Because of the poor hygiene in the hospital, unhygienic water supply for hand washing (Aiken et al., 2014), prolonged hospitalization and overcrowded patients, and lack of expert staffs favor the spread of S. aureus in hospital (Aiken et al., 2014;Ahmed et al., 2019).Besides, it is well known that wound infection usually caused by multiple-resistant S. aureus (multi-drug resistant S. aureus).So, to reduce the morbidity of surgery patients with wounds, the clinician should dispatch the wound specimen to the laboratory for culture, and sensitivity to select effective antibiotic.
The culture positivity of human samples was 70% which is similar to a previous study (Anupurba et al., 2003).The higher number of culture-positive samples were due to the lack of hygiene in the hospitals.Thus, for the prevention of nosocomial infections, the standards of hygiene in the hospitals should be improved (Chambers and DeLeo, 2009;Aiken et al., 2014).The overall prevalence of S. aureus in human specimens (40%) is similar with the findings of previous studies (Shibabaw et al., 2014;Suzuki et al., 2020) and are considered as quite high.The majority of S. aureus (42.5%) were from surgical wound swab.The higher proportion of S. aureus from the wound was also reported previously (Nwachukwu et  Antimicrobial resistance of the pathogenic bacteria is of great concern both in human and veterinary medicine worldwide and is a significant problem in the treatment of human and animal diseases caused by pathogenic S. aureus (Chambers and DeLeo, 2009;Dutta et al., 2013;Ahmed et al., 2019).S. aureus has become resistant to many commonly used antibiotics through the acquisition of antibiotic resistance genes (Foster, 2017).A major factor is the acquisition of resistance genes from other resistant bacteria (e.g., the spread of plasmidencoded multidrug transporters) and selfacquisition of antibiotic-resistant genes (e.g., overexpression of endogenous multidrug transporters) due to irrational antibiotic prescribing by physicians and surgeons, a habit of self-medication among patients in developing countries, and indiscriminate use and huge consumption of antibiotics in animal husbandry, and agriculture farming globally (Lowy, 2003;Foster, 2017).Nowadays, we have to combat these insidious enemies by finding effective therapeutic agents (Dutta et al., 2013).
In antibiotic resistance test, animal origin samples exhibited a higher resistance against penicillin which is similar with the findings of previous studies (Kurjogi and Kaliwal, 2011).The most likely reason for the higher resistance of penicillin among dairy cows might be due to the habit of indiscriminate use of antibiotics in livestock farming (Chambers and DeLeo, 2009;Mostafa and Kumar, 2012;Sutradhar et al., 2014;Biswas et al., 2014).These findings indicate that choice of penicillin for the treatment of staphylococcal disease in the animal are not a good option.This might cause failure to cure diseases of animals as well as there might be chances of transmission of antibiotic resistance bacterial genes to the human cycle through meat, milk, eggs, and other animal products (Ahmed et al., 2019).So judicious use of antibiotics for the treatment of staphylococcal diseases are suggested (Foster, 2017).Choice of alternate antibiotics, controlled use of antibiotics through strict regulations, educating the general public and health care persons (Chen et al., 2016) about hazards of antibiotics might reduce antibioticresistant microorganisms globally.
The antibiotic resistance pattern of S. aureus isolates from human samples are similar to that described previously (Naik and Teclu, 2009;Chen et al., 2016;Gurung et al., 2020).The probable reason behind this resistance phenomenon might be due to the acquisition of a plasmid and chromosome mediated resistance genes as reported by a previous study (Chamberland et al., 2001).Relatively higher resistance of S. aureus against penicillin was detected in human samples compared to animal samples which is similar to the findings of a previous study (Pandey et al., 2012).The most likely reason for this might be due to extensive use of penicillin for the therapy of human diseases caused by S. aureus (Lowy, 2003).Other factors might also be responsible for the development of penicillin resistance are the misuse of penicillin, poor quality drugs, undernutrition, compromised immunity, substandard health care facilities, inability to afford costly but more effective drugs ( (Islam et al., 2011).The present findings indicate that except penicillin, all other tested antibiotics could be used for the treatment of animal and human diseases caused by S. aureus.But every nation should undertake and implement effective surveillance programs (antibiotic stewardship programs) following standardized methodologies to find out the development of new antimicrobials resistance (Sumon et al., 2018) to prevent the spread of antimicrobialresistant S. aureus globally through humans, animals, animal products, and environment etc. (Sjölund et al., 2008;Dutta et al., 2013;Haque et al., 2020).
One of the main limitations of the present study is that the disc diffusion method cannot detect low levels of antibiotic resistance in microorganisms.Future studies should undertake to find out resistance of microbes against different antibiotics by using molecular detection techniques.But such methods are costly and requires highly experienced personnel.

Conclusion
The infections caused by S. aureus both in humans and animals could be considered as high.S. aureus isolates from both animals and humans exhibit a higher percentage of resistance against penicillin.So, care should be taken during use of penicillin for the therapy of infections caused by S. aureus in animals and humans.It is essential to find alternate antimicrobials for the treatment of diseases caused by S. aureus in both animals and humans to prevent the spread of staphylococcal resistance genes among animal health, human health, and environment.Novel technologies leading to rapid and accurate diagnosis of S. aureus infection and improved understanding of the pathogenesis and pathology of the S. aureus diseases are crucial.Along with the antibiotic approach, non-antibiotic approaches are essential for the prevention of the spread of multi-drug resistant S. aureus infections globally.

Fig. 1 (
Fig. 1 (a).Nutrient agar media showing growth of S. aureus.Fig. 1 (b).Blood agar media showing growth of S. aureus with hemolysis.Fig. 1 (c).Gram-positive spherical cells of S. aureus arranged in grape-like clusters.Fig. 1 (d).Mannitol Salt Agar media showing yellowish colour after growth of S. aureus.Fig. 1 (e).Catalase test showing gas bubbles as an indication of positive reaction for S. aureus in two of three test tubes.Fig. 1 (f).Coagulase test showing positive coagulase reaction in two of three test tubes (conversion of plasma into a soft and/or stiff gel).

Fig. 2 (
Fig. 2 (a).Arrow indicator antibiotic sensitivity disc in Mueller Hinton agar media showed Oxacillin sensitive S. aureus.Fig. 2 (b).Arrow indicator antibiotic sensitivity disc in Mueller Hinton agar media showed Oxytetracycline resistant S. aureus al., 2009; Pandey et al., 2012; Roy et al., 2017).The prevalence of S. aureus infection in clinical patients in hospital was high because S. aureus remains as commensals on the skin (Rahman et al., 2005; Chambers and DeLeo, 2009; Gurung et al., 2020).From skin, S. aureus spread to the infection site if the wound surface of the patient remains exposed.

Table 4 . Antibiogram study for S. aureus isolated from different clinical specimens of human by disc diffusion method (n = 40)
Erami et al., 2014; Neyra et al., 2014; Chen et al., 2016; Roy et al., 2017; Anueyiagu et al., 2020).The lowest percentage of animal origin S. aureus were resistant to oxacillin and the lowest percentage of human origin S. aureus were resistant to fusidic acid.The lowest resistance of animal origin S. aureus to oxacillin is due to the absence of oxacillin preparation in the local market for the therapy of animal diseases