Detection and Antimicrobial Resistance of Staphylococcus Species from Chicken, Chicken Litter, and Humans in Addis Ababa, Ethiopia

Background In veterinary medicine, three Staphylococcus species are of particular importance as primary causes of specific diseases; S. aureus (mastitis in ruminants, equine botryomycosis, and bumble foot in poultry), S. hycus (porcine exudative epidermitis), and S. intermedius (canine pyoderma). The disease conditions caused by Staphylococcus in poultry vary with site, route, and predisposing factors include wounds as a result of fighting/cannibalism, immunosuppression based on virus infection or parasite infestation, and bad husbandry conditions (overcrowding). The objectives of this study were to isolate and identify Staphylococcus spp from chicken and chicken litter and personnel at chicken farm and to determine the antimicrobial susceptibility profile of the isolates. Methods A cross-sectional study was conducted on apparently healthy chickens, farm personnel, and chicken litter at poultry farms in Addis Ababa, Ethiopia. A total of 222 samples consisting of 101 cloacal swabs, 90 tracheal swabs, 17 pooled litter swabs, 7 nasal swabs, and 7 pooled hand and boot swabs were collected from six farms and examined for the presence of Staphylococcus species. Antimicrobial resistance against 10 antimicrobial agents was also conducted following recommended standard procedures. Results Overall proportion of Staphylococcus was 64/222 (28.83%). Of the isolates, 40/64 (62.5%), 11/64 (17.2%), 3/64 (4.7%), and 10/64 (15.6%), were S. aureus, S. hycus, S. intermedius, and coagulase-negative staphylococci (CNS), respectively. Only one isolate of S. aureus was susceptible to all antimicrobials tested. Of the 10 antibiotics tested, the isolates demonstrated highest resistance against Penicillin G (96.9%) followed by Tetracycline (78.1%), and Amoxicillin and Erythromycin at the same level (65.6%). Conversely, the isolates were highly susceptible to Ciprofloxacin (95.3%) followed by Sulphamethoxazole-trimethoprim (85.9%). Out of 64 isolates, 61/64 (95.3%) were resistant to three or more antimicrobials tested. Of the isolates, 38/40 (95%) S. aureus, 10/11 (90.9%) S. hycus, 3/3 (100%) S. intermedius, and 10/10 (100%) CNS showed multidrug resistance. Conclusion This study showed a considerable proportion of Staphylococcus spp in chicken litter and farm workers with a potential source of resistant Staphylococcus species, and more importantly multidrug resistance strains. Further studies on molecular characterization of the isolates will be essential to identify the resistant genes and establish epidemiological links in the transmission dynamics of resistant Staphylococcus species between poultry and humans.


Background
Staphylococci are facultative anaerobic, non-motile grampositive cocci, which commonly form grape-like clusters [1,2]. In humans and animals, many staphylococcal species are commensals on the skin and mucosal surfaces such as the upper respiratory tract, alimentary tract, and genitourinary tract [3]. In addition to their wide distribution, staphylococci can easily spread between different animal species, and between humans and animal species. e sources of infection are mainly contaminated foods, water and equipment, carrier, and clinically infected human and animals, and environment where the animals are crowded together. Various transmission routes have been described including direct, via the hands, contact with excretions or contact with nonliving objects (fomites), ingestion of contaminated food and water, aerosols, and via vectors [4,5].
Among the staphylococci, several studies identified the most pathogenic LA-MRSA isolated from pig, veal, calf, and dairy farms and those persons with occupational contact to livestock, such as farmers, veterinarians or abattoir workers, and other persons with exposure to livestock [6]. Although poultry plays a major role in intensive animal husbandry, there are only limited studies available on the isolation of Staphylococcus from poultry and from food of poultry origin intended for human consumption. Staphylococcal infections in both animals and humans are commonly treated with antimicrobial agents, most often with β-lactam antibiotics. ese antibiotics were initially highly effective against staphylococci, but β-lactamase-producing Staphylococcus isolates emerged in the mid-1940s, and their prevalence increased dramatically within a few years [7,8]. Staphylococci organisms, especially S. aureus strains, are known to produce beta-lactamases and acquired resistance to mobile genetic elements, plasmids, and transposons which could possibly play a vital role in the emergence of multiple drug resistant [9]. In Ethiopia, few research studies have been reported in the country pertaining Staphylococcus isolation and identification from poultry and poultry farms. erefore, the objectives of this study were to isolate and identify and to assess the antimicrobial susceptibility patterns of Staphylococcus spp from chicken litter and poultry farm personnel in Addis Ababa.

Study Area.
e study was conducted in and around Addis Ababa. Addis Ababa is the capital city and administration centre for the Federal Democratic Republic of Ethiopia. Currently, there are 10 subcities "Kifle Ketemas" in Addis Ababa city administration delineated on the basis of geographical setup, population density, assets and service providers' distribution, and convenience for administration [10]. It is situated at the latitude of 9°3′ north and 38°43′ east longitudinally. It lies in the central high lands of Ethiopia at an altitude of 2400 m.a.s.l. It has an average rainfall of 1800 mm per annum. e annual average maximum and minimum temperatures were 26°C and 11°C, respectively, with an overall average of 18.7°C. Highest temperatures are reached in May. e main rainy season extends from June to September. It has a relative humidity varying from 70% to 80% during the rainy season and 40% to 50% during the dry season. Addis Ababa covers about 54,000 hectors of land with an average population of more than 3 million [11].

Study Design, Samples, and Sampling.
A cross-sectional design was used to generate the desired data from a total of 222 samples, collected randomly from apparently healthy exotic chickens. e sample types were cloacal and tracheal swabs from both layers and broilers, pooled litter swabs from each farm and nasal and pooled hand and boot swabs from farm workers.
An informed verbal consent was obtained from farm owners and workers. Cloacal and tracheal swab samples were collected into a single screw capped test tube containing 10 ml of buffered peptone water as transporting media. Each sample was labelled with necessary information, including date of sampling, type of sample, source of sample (farm), and identification of the animal. Finally, the samples were properly packed in an ice box and transported to the microbiology laboratory at the College of Veterinary Medicine and Agriculture of Addis Ababa University, Bishoftu, for bacteriological analysis.

Isolation and Identification.
e International Organization for Standardization, ISO 6888-3:2003, was employed for the isolation and identification of Staphylococcus species from swab samples. A loop full of the pre-enriched samples was streaked on a blood agar plates (BAP) enriched with 7% heparinised sheep blood and incubated at 37°C for 24-48 hours under aerobic conditions. e plates were examined for the presence of Staphylococcus colonies based on the morphological characteristics (creamy, greyish white, or yellow colonies) and haemolytic pattern. e presumed colonies were further subcultured on nutrient agar plates (NAP) and incubated at 37°C for 24-48 hours to obtain pure colonies. e pure colonies were transferred to nutrient agar slants and stored at 4°C for further biochemical and antimicrobial susceptibility tests. Identification of staphylococci and species assignment was done based on KOH test, Gram's staining, catalase test, oxidation and fermentation test, sugar fermentation (mannitol and maltose) tests, and coagulase test [12].

Antimicrobial Susceptibility Test.
A sterile swab was dipped into the standardized suspension of bacteria, and excess fluid was expressed by pressing and rotating the swab firmly against the inside of the tube. e swab was streaked in three directions and continuously brushed over the Mueller Hinton agar, and inoculated plates were allowed to stand for 3-5 minutes. e discs were placed onto the agar surface using sterile forceps and gently pressed with the point of a sterile forceps to ensure complete contact with the agar surface, and the plates were incubated aerobically at 37°C for 16 hours-18 hours for all disc except for Vancomycin 24 hours. Inhibition zone diameters were measured, and values obtained from the Clinical laboratory standard institute [13].

Data Management and Analysis.
e generated data were stored in Microsoft Excel and analysed using SPSS version 20 software. Descriptive statistics such as percentage, the proportion, and frequency distributions were applied to compute some of the data. Chi-square (χ 2 ) was used to compare sample source and types.
ere was no significant association between isolation rate and housing system (P � 0.103) or sample source (P � 0.052) ( Table 1).
Among the Staphylococcus species, S. aureus was isolated from all sample sources, broiler (17.9%), layer (17.5%), litter (23.5%), and personnel (14.3%). Both S. hycus and CNS with the same result (6.7%) were isolated from broilers; S. intermedius was only isolated from broilers (2.2%). ere was no statistically significant difference in Staphylococcus species isolated and identified between different sample sources (P value ≥0.05 in all species) ( Table 2). All staphylococcal species were isolated from different sample types, but none of them were identified from the pooled hand and boot swabs of farm attendants. S. aureus was the highest (28.6%) in nasal swabs and lowest (12.9%) in cloacal swabs. S. aureus 4 (23.5%). S. hycus 2 (21.8%), and CNS 1 (5.9%) were isolated from litter, but S. intermedius was not. S. intermedius was only isolated from cloacal swab, 3/101 (3%). ere was no statistically significant association observed in Staphylococcus species isolated from different sample types except CNS (P � 0.05) ( Table 2).
All isolates (64) of Staphylococcus were tested for susceptibility test to 10 antimicrobial discs. e comparative efficacies of antimicrobials used indicate that CIP and SXT were the most effective antibiotics with susceptibility percentage of 95.3% and 85.9%, respectively. Conversely, P and TE have shown the poorest efficacy (susceptibility) or high resistance against staphylococcal isolates with 96.9% and 78.1%, respectively, ( Table 3).
Out of 64 Staphylococcal isolates, only one (1.56%) isolate (S. aureus) was susceptible to all tested antimicrobial agents, while two (3.13%) were found to be monodrug resistant. Multidrug resistance to three or more antimicrobials was observed in 61 (95.31%) of all isolates (Table 5).

Discussion
Staphylococcus spp. are significant bacteria in the aetiology of avian diseases, but little is known about the bacterial presence in the poultry environment such as in poultry litter and in the poultry house air as reported by Saleh et al. [14]. e modern poultry industry can produce market-ready broiler chickens in less than six weeks. is accomplishment has been achieved through genetic selection, improved feeding, and keen health management practices including the usage of antibiotics as therapeutic agents to treat bacterial diseases in intensive farming systems. Resistance to frequently used antibiotics has been observed in bacteria present in poultry since the introduction of these antimicrobial agents in poultry [15].   Key: CNS � coagulase negative Staphylococcus; SA � S. aureus; SH � S. hycus; SI � S. intermedius; χ 2 � chi-square, P � P value, CI � confidence interval, and C � cage type housing system; L � litter-type housing system; P � personnel, Ly � layer, B � broiler, and Cl � loacal swab; L � litter swab and T � tracheal swab; N � nasal swab; HB � hand and boot swab.

Veterinary Medicine International
Out of a total of 222 samples, 64 (28.8%) were found to be positive for Staphylococcus species which is lower than the finding of Modestas et al. [16] who reported 71% Staphylococcal species from poultry products in Kaunas, Lithuania, but higher than 15.2% Staphylococcal species reported by Masdooq et al. [17] from pathogenic bacteria associated with respiratory diseases of poultry in Nigeria.
Of the total isolates, it was found that 62.5% of them were S. aureus which is higher than the previous work of [18] that reported the isolation of S. aureus (23.53%) from yolk sac infection (omphalitis) in Kombolcha poultry farm, Ethiopia, and [19] who reported 20.5% S. aureus from chicken in Jos, Nigeria. Moreover, the present finding revealed the prevalence of S. aureus (62.5%), S. hycus (17.2%) and, S. intermedius (4.7%) was nearly the same with 2%, and CNS (15.6%). In the previous study in Morocco, it was reported that S. aureus (40%), Staphylococcus intermedius (2%), and Staphylococcus hyicus (4%) were much lower than 54% which were (i.e., 40%, 4%, 2%, and 54%) the findings of [20] in Morocco. Other observation was also reported by [21]    with a prevalence of 46% S. aureus from poultry products in Egypt which was of course lower than the present study value.
Of the samples collected from broiler, layer, litter, and farm attendants, 45 (33.58%), 10 (17.54%), 7 (41.18%), and 2 (14.29%), respectively, were found to be positive for Staphylococcus species. Similarly, Saleh et al. [14] reported that the prevalence of Staphylococcus in broiler was 32% which is comparable with our finding (33.58%) in broilers. Moreover, they have detected 35% Staphylococcus species in layers which is much higher than the present result (17.54%). In our study, the S. aureus isolated from broiler and layers was 17.9% and 17.5%, respectively, which is much lower than the finding of [22] who reported 50.89% S. aureus from broilers in Iraq. Similarly, the present finding of S. aureus from litter (23.5%) was lower than the finding of [23] who reported 33.3% in Khartoum state. In the present study, the detection of S. aureus from nasal swabs (28.8%) is higher than the finding of [24] who reported 3.61% from nasal swabs of poultry farm workers in Iran; however, the detection of the isolate from tracheal swab (23.3%) was much lower than the finding of [25] that reported 85% of S. aureus from tracheal swab. e variation in detection of the isolates may emanate from several factors; the variation in the diagnostic capability of the laboratories may vary from country to country, geographical variations, variation in breeds, management practices, etc.
In this study, 95.0% S. aureus isolates were found to be resistant to Penicillin G. A previous study also revealed a similar finding where 96.7% S. aureus were found to be resistant to Penicillin G [26] but slightly lower than 100% resistance reported by Otalu et al. [27]. In this study, 100% resistance towards Penicillin G is recorded by S. hycus, and CNS showed the highest resistance.
is finding is in consistent with that of [20] who reported similar results from milk and whey in Morocco. us, the results indicate that the majority of antimicrobial resistance in S. aureus and CNS isolates could be due to the production of β-lactamases and may carry the mecA chromosomal gene responsible for the production of the altered penicillin binding protein PBP-2a as suggested by [15].
Of the total isolates (64) subjected to the antimicrobial susceptibility test, 61/64 (95.3%) have developed multidrug resistance (resistant to three and more than three antimicrobials). High prevalence of multidrug resistance among isolates in the present study clearly indicated the excessive or   (1)  6 Veterinary Medicine International inappropriate use of antibiotics. is may be connected to the fact that antimicrobial use in commercial poultry settings is more likely to be regulated than in small holder farms. In addition, veterinarians and poultry farmers generally use these antimicrobials as prophylaxes, growth promoters, or inaccurate dosages given to sick flocks by unqualified personnel may likely result a high level of resistance that was reported by [25] done on antimicrobial resistant Staphylococcus from chickens in Maiduguri, Nigeria, and it is in agreement with present finding of multi-drug resistance.

Conclusion
e results of the current study showed that Staphylococcus is one of the organisms that are widely distributed in poultry farms in the study area. In this study, a total of 222 samples were examined and processed, and 64 (28.83%) Staphylococci were isolated and of which S. aureus was the most dominant one, 40 (62.5%), even though little or no comparative report in Ethiopia at all. e antimicrobial susceptibility test of the Staphylococcus isolates in this study showed that 61/64 (95.31%) isolates, almost all were multidrug resistance (three and more than three antimicrobials tested), and this makes an alarming cause for further studies.
erefore, further studies should be conducted on a large scale to find the association between source of infection and prevalence to find out the possible source of contamination with Staphylococcus species, and antimicrobial susceptibility test should be carried out at regular intervals to determine the development of resistance against the most commonly applied antibiotics.
Data Availability e data can be obtained from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interests.

Authors' Contributions
Fufa Abunna conceived and designed the experiments, and contributed reagents and materials. Biyansa Adugna performed the experiments, analysed and interpreted the data, and wrote the paper. Hika Waktole performed experiments. Takele B. Tufa conceived and designed the experiments, performed the experiments, and contributed reagents and materials. Dinka Ayana conceived and designed experiments. Fanta D. Gutema wrote and edited the paper. Reta D. Abdi conceived and designed the experiments, performed the experiments, analysed and interpreted the data, and contributed reagents, materials, analysis tools, or data.