Detection and Antimicrobial Resistance of Staphylococcus Spp From Chicken, Litter and Humans in Addis Ababa, Ethiopia

Background: In veterinary medicine, three Staphylococcus species are of particular importance as a primary cause of specic 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 the site, the route and predisposing factors include wounds as a result of ghting/cannibalism, immunosuppression based on virus infections or parasite infestations, and bad husbandry conditions (overcrowding). Besides their role as colonizer or pathogen in different hosts, Staphylococcus, which colonizes food-producing animals, can contaminate carcasses during slaughter and play a role as contaminant in the subsequent manufacturing process in food of animal origin, such as pork, beef, veal, milk, poultry meat or poultry meat Methods: A cross-sectional study was conducted on apparently healthy chicken, farm personnel and litter at chicken farms in Addis Ababa, Ethiopia from March 2015 to May 2015. The objectives of this study were to isolate and identify Staphylococcus spp from chicken, litter and personnel at chicken farm; and to determine the antimicrobial susceptibility prole of the isolates. A total of 222 samples consisting of 101 cloacal swabs, 90 tracheal swabs, 17 pooled litter swabs, 7 nasal swabs and 7 pooled hands and boot swabs were collected from six farms and examined for the presence of Staphylococcus species and antimicrobial resistance against 10 antimicrobial agents following recommended standard procedures. Results: The result showed that the 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 CNS, respectively. Only one isolate of S. aureus


Background
Staphylococci are facultative anaerobic, Gram-positive, and immotile cocci, which commonly form grapelike clusters. (Selbitz 2007;Todar, 2008). In humans and animals, many staphylococcal species are commensals on skin and, mucosal surfaces such as upper respiratory tract, alimentary tract and genitourinary tract (Aarestrup and Schwarz, 2006;Weese, 2010). In addition to their wide distribution, staphylococci can easily spread between different animal species, and also between humans and animal species. The 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, often via the hands, contact with excretions or contact with non-living objects (fomites), ingestion of contaminated food and water, aerosol and via vectors (Cuny et al., 2010;Ferreira et al., 2011) Among the Staphylococci several studies identi ed the most pathogenic one LA-MRSA isolates 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 (Köck et. al. 2013).
Although poultry plays a major role in intensive animal husbandry, there are only limited studies available on isolation of Staphylococcus from poultry and also 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. These 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. (Liu et al., 2012;Geenen et al., 2013).
Staphylococci organism specially S. aureus strains are known to produce beta-lactamases and a modi ed PBP, expression of an alternative penicillin-binding protein, called PBP2a or PBP2' and acquired resistance by mobile genetic elements, in particular plasmids and transposons possibly contributing to the emergence of multiple drug resistant (McCallum et al., 2010).
In Ethiopia, investigations on bacterial isolation and identi cation from poultry and poultry farms in general and isolation and identi cation of Staphylococcus species in particular have received little attention. To the best of our knowledge, no signi cant research has been reported in the country pertaining Staphylococcus isolation and identi cation from poultry and poultry farms. Therefore, 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 farms personnel in Addis Ababa.

Study Area
The study was conducted from October 2014 to May, 2015 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 sub-cities "Ki e Ketemas" in Addis Ababa city administration delineated on the basis of geographical set up, population density, asset and service providers' distribution and convenience for administration (AACA, 2004). It is situated at 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. The annual average maximum and minimum temperature is 26 °C and 11 °C, respectively; with an overall average of 18.7 °C. Highest temperatures are reached in May. The main rainy season extends from June to September. It has a relative humidity varying from 70-80% during rainy season and 40-50% during the dry season. Addis Ababa covers about 54,000 hector of land with an average population of more than 3 million (NMSA, 2007).

Study Population
The study population constituted apparently healthy chicken (i.e. broiler and layer) in chicken farms, farm environment (litter) and farm attendants.

Study Design
A cross-sectional design was used to generate the desired data.

Samples and Sampling
A total of 222 samples were collected randomly from apparently healthy exotic chicken from the farms.
The sample types were cloacal and tracheal swabs from both layer and broiler, pooled litter swabs from selected farms and nasal and pooled hand and boot swabs from farm workers.
An informed verbal consent was obtained from farm owners and farm workers. Cloacal and tracheal swabs were taken from each selected poultry farms and after handling the selected chicken properly. Brie y, one person held the chicken rmly while another person inserted the swab slowly "screwing" it into the tracheal cavity or cloaca of the bird. For every swab sample, (BPW) was used. Prior to sampling, swab tips were moistened in the buffered peptone water (mainly for litter swab) and swabbed by rotating and rubbing against the sampled surface several times and kept in a sterile test tube lled with 5 ml of BPW. All samples were collected aseptically using disposable gloves to avoid contamination. Each sample was labelled with necessary information, including date of sampling, type of sample, source of sample (farm) and identi cation of the animal labelled with permanent marker. At all levels of sampling, the samples were placed in the rack for easy handling and held in an ice box, properly packed and kept cold. Finally it was transported to the Microbiology laboratory at the College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu for bacteriological analysis

Isolation and Identi cation
The International Organization for Standardization, ISO 6888-3:2003 was employed for the isolation and identi cation of Staphylococcus species from swab samples. The swabbed samples which were kept in a tube containing BPW were put overnight in a refrigerator. A loop full of the pre-enriched samples various dilutions were streaked on blood agar plate (BAP) enriched with 7% heparinised sheep blood and incubated at 37 o c for 24-48 hours under aerobic conditions. The plates were examined for the presence of Staphylococcus colonies based on the morphological characteristics (creamy, greyish, white or yellow colonies) and haemolytic pattern. The presumed colonies were further sub-cultured on Nutrient agar Plate (NAP) and incubated at 37 o c for 24-48 hours to obtain pure colonies). The pure colonies were transferred to nutrient slants and stored at 4 o c for further biochemical and antimicrobial susceptibility tests.
Identi cation of staphylococci organisms and species assignment were done based on KOH test, gram's staining, catalase test, oxidation and fermentation test, sugar fermentation (mannitol and maltose) tests and coagulase test.

Antimicrobial Susceptibility Test
Antimicrobial susceptibility pattern of the isolates were determined against10 antimicrobial agents using the Kirby-Bauer disc diffusion method. Brie y, sterile cotton swab was used to streak the bacterial colonies on the surface of Mueller Hinton agar plates and were allowed to dry for 15 minutes before applying the discs Filter paper disks containing designated amount of the impregnated antimicrobial drugs obtained from commercial supply rms were gently and rmly placed on the agar plates using sterile toothed forceps. All the discs were gently pressed with forceps to insure complete contact with the agar surface. The discs were placed 1.5 cm away from the edge of the plates and they were 3 cm apart from each other. The plates were then incubated inverted aerobically at 37 °C for 18-24 hours. The diameter of the inhibition zones around disk were measured using calliper on the back of the plates and the isolates were interpreted as susceptible, intermediate and resistant according to guidelines of (CLSI,

2013). Data Management And Analysis
The generated data were stored in Microsoft excel and analysed using SPSS 20 version software. Descriptive statistics such as percentage, proportion and frequency distributions were applied to compute some of the data. The Pearson's chi-square (x 2 ) test was used to assess the differences of proportion of Staphylococcus and Staphylococcus species among the sample sources and types. The difference was considered statistically signi cant when the P-value was less than 0.05. Proportional distribution of Staphylococcus isolates in housing system and sample source From two poultry housing system, cage type and deep litter system, 4/28 (15.69%), and 58/194 (31.71%), were positive for Staphylococcus, respectively. For samples examined from layers (Bo vans Brown) and broilers (White Leg Horne), 10/57 (17.54%) and 45/134 (33.58%) were positive for Staphylococcus, respectively, but the highest percentage 7/17 (41.18%) was isolated from litter and the lowest percentage 2/4 (14.29%) was isolated from farm workers, however there was no statistically signi cant difference in isolation of Staphylococcus between different housing system (P-value = 0.103) and sample source (Pvalue = 0.052) ( Table 1).

Proportional distribution of Staphylococcus isolates in sample type
Of the total 64/222 (28.8%) Staphylococcus isolates, 33/101 (32.7%), 22/90 (24.44%), 7/17 (41.2%) and 2/7 (28.6%), from cloacal swab, tracheal swab, pooled litter swab and nasal swab of farm attendants, were positive for Staphylococcus, respectively, but no isolate was found from pooled hand and boot swab of farm attendants 0/7 (0%). There was no statistically signi cant difference in Staphylococcus isolation between different sample type (P-value = 0.225) ( Table 1).  Table 2). All the staphylococcal species were isolated from different sample types, but none of them identi ed from pooled hand and boot swab of farm attendants. S. aureus was highest (28.6%) in nasal swab and lowest (12.9%) in cloacal swab. S. aureus 4(23.5%). S. hycus 2(21.8%) and CNS 1(5.9%) were isolated from litter, but S. intermiedius not. S. intermidius was only isolated from cloacal swab, 3/101 (3%). There was no statistically signi cant difference in Staphylococcus species isolated from different sample types except CNS (p = 0.05) ( Table 2).    intermedius showed greater resistance (100%) to P, AML, TE and S, but greater susceptibility (100%) was seen in VA. Moreover, CNS has showed greater resistance (100%) to P and greater susceptibility (100%) to CIP (Table 4).     Out of 222 samples, 18.01%, 4.95%, 1.35% and 4.5% of S. aureus, S. hycus, S, intermedius and CNS were isolated, respectively. Other ndings was reported by (El-Jake et al 2008) with 8%, 2% and 0% of S. aureus, S, intermedius and S. hycus, respectively which were lower than the present ndings. The prevalence of Staphylococcus between farm levels showed slightly high percentage of variation in distribution, and the signi cance difference (P < 0.05) between the farms These variations might be due to difference in sample size, geographical location and type of bird; and it could also be related with poor hygienic and sanitation status of the difference of the farms. The water and feed troughs were not properly washed and create favourable environment for the growth and proliferation of bacterial organisms when subsequently re lled without cleaning as reported by Rany Roy et al. (2012). On the basis of individual sample types (cloacal swab, tracheal swab, nasal swab, pooed litter swab and pooled hand and boot swab) were used for isolation and identi cation of Staphylococcus and Staphylococcal species in the current study. The proportional distribution of Staphylococcus from sample types is 33(32.67%), 22 (24.44%), 7 (41.18%) and 2 (28.57%) from cloacal swab, tracheal swab, pooled litter swab and nasal swab, were positive for Staphylococcus species, respectively. From the isolates, CNS was isolated only from cloacal swab (8.9%) and pooled litter swab (5.9%); S. hycus was isolated from pooled litter swab (11.8%), cloacal swab (7.9%) and tracheal swab (1.1%); S. intermidius was only isolated from cloacal swab (3%). S. aureus was almost isolated from all sample types except pooled hand and boot swab of farm workers. Its prevalence was almost similar in pooled litter swab (23.5%) and tracheal swab (23.3%) but vary in others which is highest (28.6%) in nasal swab of farm workers and lowest (12.9%) in cloacal swab. But the present nding from nasal swab ( Bendahou et al. (2008) who reports similar result of resistance from these strains obtained from milk and whey in Morocco. Thus, the results indicate that the majority of antimicrobial resistance in S. aureus and CNS isolates could be due to production of β-lactamases and may carry the mecA chromosomal gene responsible for production of the altered penicillin binding protein PBP-2a as suggested by Mamza et al.

Proportional distribution of Staphylococcus species in farm
(2010).
Of the total isolates (64) subjected to antimicrobial susceptibility test, 61/64 (95.3%) have developed multidrug resistance (resistant to three and more than three antimicrobials). High prevalence of multi drug resistance among isolates in the present study clearly indicated the excessive or inappropriate use of antibiotics. This 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 ocks by unquali ed personnel may likely result high level of resistance that was reported by Suleiman et al. (2013) done on antimicrobial resistant Staphylococcus from chickens in Maiduguri, Nigeria, and it is in agreement with present nding of multi drug resistance.

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The result of the current study showed that Staphylococcus is one of the organisms that widely distributed in poultry farm 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. The antimicrobial susceptibility test of the Staphylococcus isolates in this study showed that 61/64 (95.31%) isolates, almost all were multi-drug resistance (three and more than three antimicrobials tested) and this makes an alarming cause for further study. Therefore, further studies should be conducted on large scale to nd the association between source of infection and prevalence to nd out the possible source of contamination with Staphylococcus species and antimicrobial susceptibility test should be carried out at regular intervals to nd out the development of resistance against the most commonly applied antibiotics.