Research Article

 

 

Antimicrobial Susceptibility Profile of Staphylococcus aureus Isolates Recovered from Various Animal Species

 

Faiza Habib1, Kanwar Kumar Malhi1*, Asghar Ali Kamboh1, Rahmatullah Rind1, Rehana Burriro2

1Department of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70060, Pakistan; 2Department of Veterinary Pharmacology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70060, Pakistan.

Editor | Sanjay Kumar Singh, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly (UP), India.

Received | October 18, 2015; Revised | November 20, 2015; Accepted | November 22, 2015; Published | December 09, 2015

*Correspondence | Kanwar Kumar Malhi, Sindh Agriculture University, Tandojam, Pakistan; Email: kanwar103@gmail.com

Citation | Habib F, Malhi KK, Kamboh AA, Rind R, Burriro R (2015). Antimicrobial susceptibility profile of Staphylococcus aureus isolates recovered from various animal species. J. Anim. Health Prod. 3(4): 99-103.

DOI | http://dx.doi.org/10.14737/journal.jahp/2015/3.4.99.103

ISSN | 2308–2801

Copyright © 2015 Habib et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Staphylococcus aureus (S. aureus) is known as anaerobic, Gram negative cocci that commonly causes infections in humans and food animals. It is found as a normal bacterial flora of skin and mucosal surfaces of the respiratory, upper alimentary and urogenital tract. It is known as one of the most prevalent and clinically significant pathogens worldwide. It causes a variety of illnesses ranging from superficial skin eruptions to life threatening infections with bacteremia, endocarditis, pneumonia and toxic shock syndrome (Rubin et al., 2011). S. aureus is a major cause of morbidity and causes significant economic losses in farm animals. In poultry, it is a main cause of septic arthritis, septicemia and limb infections (Nazia et al., 2015); in dairy animals it is responsible to cause clinical and sub-clinical mastitis (Tyagi et al., 2013); in horses it is reported as causative agent of dermatitis and septic arthritis (Weese et al., 2006); while in dogs it causes otitis externa and urinary tract infections (Silva et al., 2001). It has also been reported in inflammatory conditions of bones and joints, cutaneous lesions, septicemia, pneumonia, toxic shock syndrome, meningitis and renal impairments, besides food poisoning (Pereira et al., 2011). Since, methicillin–resistant Staphylococcus aureus (MRSA) was first identified in 1961, it has become the most common cause of nosocomial and community infections worldwide (Deresinski, 2005).

 

A wide array of antibacterial agents are being used to treat bacterial infections including those caused by S. aureus. The indiscriminate use of antibiotics particularly in livestock industry as prophylactic and growth promoting agents results the emergence of antibiotic resistant bacteria (Ansari et al., 2014). Resistance to commonly used antimicrobials is frequently encountered with S. aureus. A shift in resistant or sensitivity pattern from time to time in a particular area is well established. Moreover, resistance level is largely variable from area to area (Rubin et al., 2011). Therefore for rationale use of antibiotics there is a great need to explore the antibiotic sensitivity profile of important pathogens on periodical basis. The objective of this study was to determine in vitro antimicrobial sensitivity profile of an archived collection of S. aureus isolates recovered from different animal species in Hyderabad, Pakistan. This study, additionally will illuminate the concepts of host-microbe interaction for development of antimicrobial resistance.

 

MATERIALS AND METHODS

 

Bacterial Isolates

Staphylococcus aureus isolates (n=80) were recovered from adult animal species including sheep, goat, buffalo, camel, horses, cattle, dog and human. These were isolated from various types of samples such as blood, pus, milk, injuries, and surgical and non-surgical wounds. The samples were collected from Hyderabad, a second largest city of Sindh province of Pakistan. All Samples were collected in sterilized bijoux bottles under strict aseptic conditions and transported to the laboratory in a refrigerated container. These were streaked onto nutrient agar, MacConkey’s agar and blood agar with sterilized platinum loop and incubated at 37°C for overnight. The detailed identification of the S. aureus isolates were done as described by Malik (1986). Streak plate method was adopted to obtain distinct colonies. S. aureus were identified on the basis of cultural characteristics, colony morphology, Gram’s staining behavior, and biochemical tests including fermentation of sugars and production of coagulase. After identification, isolates were snap frozen in skim milk at –80°C until analyzed for in vitro antimicrobial susceptibility.

 

In Vitro Antimicrobial Susceptibility Testing

In vitro antimicrobial susceptibility testing for eleven different antibiotics was performed using the disk diffusion method as described by NCCLS (1993). For this purpose, Muller Hinton agar (Difco laboratories, USA) was prepared and dried by incubating at 37°C for 30 minutes. Commercially prepared discs (Becton, Dickinson, Cockeysville, MD, USA) for the following antimicrobials were used: ampicillin (10µg), amikacin (30µg), chloramphenicol (30µg), sulfanilamide (30µg), erythromycin (15µg), gentamicin (10µg), kanamycin (30µg), neomycin (30µg), ofloxacin (5µg), penicillin G (10 IU) and tetracycline (30µg). Drug dosages were chosen as described by Abo-State et al. (2012). Discs were placed over the surface of agar plate with the help of disc dispenser and slightly pressed with sterile forceps to make it adhere to the surface of the medium. The plates were closed, wrapped in aluminum foil, inverted (medium up and disc downward) and incubated for 24h at 37°C. The zones of inhibition for antimicrobial discs were observed and recorded with annotations.

 

RESULTS

 

Susceptibility Profile of S. aureus Isolated from Buffalo

During present study, buffalo isolates of S. aureus were found highly sensitive to ofloxacin, erythromycin and chloramphenicol and their efficacy was recorded as 86.67,86.67 and 80.00% respectively (Table 1).Whereas, gentamicin and amikacin were found less effective against S. aureus as 60.00 and 53.33 % respectively. However, ampicillin and sulfanilamide showed complete resistance (0% sensitive) against the isolates.

 

Susceptibility profile of S. aureus isolated from cattle

As shown in Table 1, the erythromycin and chloramphenicol were found as most effective drugs against the S. aureus isolates of cattle, and their efficacy was recorded as 86.67 and 80.00% respectively. While quite effective antibiotics against S. aureus recorded were gentamicin, ofloxacin and amikacin and their levels of efficacy against the organism recorded were 60.00, 60.00 and 53.33% respectively. Ampicillin and sulfanilamide were found as a least effective drug (0% sensitive) against the isolates of cattle.

 

Susceptibility Profile of S. aureus Isolated from Camel

S. aureus isolated from the samples of camels was tested for susceptibility to various antibiotics and found highly sensitive (100.00%) to tetracycline (Table 1). Whereas, the second most highly effective drugs against the organism noted were ofloxacin, neomycin, kanamycin and amikacin, and their efficacy was recorded as 85.70%. Overall, camel isolates showed highest sensitivity for all the antimicrobial agents as compared to isolates of other animal species.

 

Susceptibility Profile of S. aureus Isolated from Goat

The susceptibility to various antibiotics against S. aureus isolated from goats was analyzed and results were summarized in Table 1. It was found that highly effective drugs against the isolate were: erythromycin (75.00%), gentamicin (75.00%), neomycin (70.00%) and penicillin G (60.00%). However, quite effective antibiotics against S. aureus noted were kanamycin (50.00%) and chloramphenicol (45.00%).

 

Susceptibility Profile of S. aureus Isolated from Sheep

As shown in 1 Table, sheep isolates of S. aureus were highly sensitive to ofloxacin, tetracycline, gentamicin, ampicillin,

 

Table 1: The in vitro antimicrobial sensitivity (%) of Staphylococcus aureus isolated from different animal species

Animal species	Antimicrobial agents*											Overall
	Amp	Ami	Chl	Sul	Ery	Gen	Kan	Neo	Pen G	Tet	Ofl
Buffalo	0.00	53.33	80.00	0.00	86.67	60.00	33.33	46.67	33.33	46.67	86.67	526.63
Cattle	0.00	53.33	80.00	0.00	86.67	60.00	33.33	46.67	46.67	46.67	60.00	513.30
Camel	71.40	85.70	57.00	0.00	42.80	78.50	85.70	85.70	0.00	100.00	85.70	692.50
Goat	0.00	0.00	45.00	0.00	75.00	75.00	50.00	70.00	60.00	33.33	0.00	408.33
Sheep	61.50	46.10	3.84	0.00	50.00	96.10	23.00	3.84	57.00	73.00	100.00	514.38
Dog	0.00	100.00	100.00	66.67	75.00	0.00	43.75	0.00	33.33	0.00	86.67	505.40
Horse	0.00	66.67	100.00	66.67	73.33	57.00	86.67	86.67	93.33	46.67	0.00	676.96
Poultry	18.75	43.75	50.00	18.75	56.25	33.33	62.50	43.75	33.33	33.33	0.00	393.74
Human	68.70	56.20	0.00	43.75	62.50	81.25	75.00	0.00	33.33	75.00	81.25	576.98
Overall	220.35	505.07	515.84	195.82	608.20	541.18	493.27	383.27	390.31	454.64	500.27

 

Amp: Ampicillin; Ami: Amikacin; Chl: Chloramphenicol; Sul: Sulfanilamide; Ery: Erythromycin; Gen: Gentamicin; Kan: Kanamycin; Neo: Neomycin; Pen G: Penicillin G; Tet: Tetracycline; Ofl: Ofloxacin

 

penicillin G and erythromycin, and their susceptibility levels were recorded as 100.00, 73.00, 96.10, 61.50, 57.00 and 50.00% respectively. However, quite effective antibiotic against S. aureus noted was amikacin (46.10%), whereas, chloramphenicol and neomycin were found as weakly effective (3.84% sensitivity) against the sheep isolates (Table 1).

 

Susceptibility Profile of S. aureus Isolated from Dog

The antibiotics, chloramphenicol and amikacin were noted as the highly effective drugs against the isolates of dog and their sensitivity level was recorded as 100.00% (Table 1). Similarly, the efficacy of ofloxacin, erythromycin, sulfanilamide, kanamycin and Penicillin G were recorded as 86.67, 75.00, 66.67, 43.75 and33.33%%, respectively. However, dog isolates of S. aureus was found 100.00% resistant to ampicillin, gentamicin, neomycin and tetracycline.

 

Susceptibility Profile of S. aureus Isolated from Horses

The bacterial organisms, S. aureus isolated from horses were tested for its susceptibility to various antibiotics and results were presented in Table 1. Chloramphenicol and penicillin G were the most effective drugs i.e., highly susceptible and their susceptibility levels were recorded as 100.00 and 93.33% respectively. While, quite effective antibiotics against S. aureus isolates of horses noted were kanamycin (86.67%), neomycin (86.67%), erythromycin (73.33%), sulfanilamide (66.67%), amikacin (66.67%), gentamicin (57.00%) and tetracycline (46.67%). Overall, after camel, the horse isolates were found most sensitive for all the antimicrobials tested as compared to isolates of other origins.

 

Susceptibility Profile of S. aureus Isolated from Poultry Birds

Overall, S. aureus isolates of poultry were found least sensitive for the antimicrobial agents (Table 1). Kanamycin, erythromycin and chloramphenicol were only the antimicrobial agents that showed ≥ 50% sensitivity for the S. aureus isolates. However, ofloxacin showed no response (0% sensitivity) for the bacterial organisms.

 

Susceptibility Profile of S. aureus Isolated from Human Being

Table 1 indicates that, the S. aureus isolates of human being was highly susceptible to ofloxacin (81.25%), gentamicin (81.25%), tetracycline (75.00%), and kanamycin (75.00%). While, ampicillin (68.70%), erythromycin (62.50%), amikacin (56.20%) and sulfanilamide (43.75%) were noted as moderately effective antibiotics against bacterial isolates.

 

Discussion

 

A number of pharmacokinetic studies have been performed for S. aureus isolated from different animal species, however, to the best of our knowledge this is a first report from Pakistan about an archived collection of S. aureus isolated from almost all farm animal species. During present study isolates of S.aureus were recovered from different animal species including sheep, goat, buffalo, camel, horses, cattle, dog and human. These were isolated from various types of samples such as blood, pus, milk, injuries, surgical and non-surgical wounds. Eleven different antimicrobial agents i.e., ampicillin (10µg), amikacin (30µg), chloramphenicol (30µg), sulfanilamide (30µg), erythromycin (15µg), gentamicin (10µg), kanamycin (30µg), neomycin (30µg), ofloxacin (5µg), penicillin G (10 IU) and tetracycline (30µg) were used to analyze the sensitivity pattern of various S. aureus isolates. Results indicated that sulfanilamide and ampicillin showed least sensitivity as compared to other antimicrobials. Similar findings were reported by Matanović et al. (2012) and Alian et al. (2012). Ampicillin is a beta-lactam antibiotic and evidences have suggested that resistance to beta-lactamase sensitive penicillin is extensive among S. aureus regardless of animal origin (Kumar et al., 2013). A recent study have also indicated the high (90.90%) resistance profile of S. aureus strains isolated from cattle, goat and sheep for penicillin (Mai-siyama et al., 2014). Sulfonamides are most common antimicrobial agents used in veterinary practices, particularly in developing countries where non judicial uses of antibiotics are very common. It results the development of resistance for sulfonamides and trimethoprim for most of pathogenic bacteria of livestock animals including S. aureus. Plasmid pWA2 has been known to cause sulfonamide resistance in S. aureus (Then, 1989).Moreover, when little doses of antibiotics were used against microbes, they prevent the growth of susceptible bacteria and leave a small number of resistance bacteria, which propagate efficiently (Farzana et al., 2004).

 

Adamu et al. (2010) isolated Staphylococcus aureus from apparently healthy humans, sheep, goats and cattle. All isolates were tested against 13 antimicrobial agents. Of these, gentamycin, norfloxacin, tetracycline, streptomycin and erythromycin showed the highest activity against S. aureus while cefotaxime, ceftazimide and ampiclox showed least activity. The authors concluded that gentamycin, norfloxacin, tetracycline, streptomycin, and erythromycin are the drugs of choice against the infections caused by S. aureus. Likewise, Parmar et al. (2014) carried out in vitro antimicrobial sensitivity assay of the 35 isolates of S. aureus from man, animal and environment origin, and showed the resistance pattern ranging from 14.29 to 60.00%. Tetracycline was found to be the most effective drug (85.72 %) followed by enrofloxacin (71.43 %), ampicillin (71.43 %), gentamicin (71.43 %), ciprofloxacin (48.57 %), co-trimaxazole (48.43 %) and furazolidone (40.00 %). The considerable difference in the susceptibility of the bacterial species to various antibiotics between the present study and that of other work might be due to host species because the host species could play role to increase or decrease the susceptibility of the microorganism to different drugs (Cameron, et al., 2011).

 

It has been hypothesized that host-pathogen interactions also play a major role in progression of antimicrobial resistance among infectious agents. In order to illuminate these connections research has focused on toxins production, release of extracellular virulence products, adherence to biotic and abiotic surfaces, and phagocytosis. Moreover, functional genomics research have recognized a number of cascade pathways responsible for host-microbe interaction. Some of those have been linked with development of resistance in microbial community for antimicrobial agents (Cameron, et al., 2011; Watkins et al., 2012). Our present results also reflected a probable role of host species in susceptibility/resistance pattern of S. aureus. As, poultry isolates showed least, while camel and horse isolates showed most response for the antimicrobial agents in present study. In agreement to our results, a recent study in The Netherlands have reported that equine isolates were predominantly susceptible to most antimicrobial agents tested, however the human isolates showed least sensitivity. They concluded that no methicillin-resistant S. aureus was present in healthy horses whereas methicillin-resistant coagulase negative Staphylococci were commonly present (Busscher et al., 2006).

 

Conclusions

 

The results were found highly varied for sensitivity pattern of S. aureus isolates of different origins. Ampicillin and sulfanilamide showed least sensitivity as compared to other antimicrobials. However, poultry isolates showed least, while camel and horse isolates showed most response for the antimicrobial agents. These results indicates the probable host-microbe interaction (as also been suggested by previous workers) and also potential role of host in progression of resistance in bacteria against antimicrobial agents. Further studies are warranted to elucidate the exact phenomenon behind the variations in antimicrobial susceptibility of S. aureus isolates of different animal origin.

 

Conflict of Interest

 

The authors declare no conflict of interest.

 

Authors’ contribution

 

This work was a part of M. Phil project of first author Fazia Habib. While, Asghar Ali Kamboh and Rahmatullah Rind were the mentors of her project. While, Rehana Burriro and Kanwar Kumar Malhi equally helped in writing and revision of this manuscript.

 

Acknowledgement

 

The authors highly acknowledged the Central Veterinary Diagnostic Laboratory (CVDL) Tando Jam for providing research facilities to carry out some part of this work.

 

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