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Isolation, DNase-cross-Coagulase test and antimicrobial resistance test on Staphylococcus along beef abattoir line in Addis Ababa Ethiopia

Staphylococci are responsible for foodborne infection and intoxication with the spread of antibiotic resistance. The aims of the study were to investigate beef abattoir line contamination with Staphylococcal , to evaluate DNase test for alternative of the tube coagulase test, and to assess isolates drug resistance in Ethiopia. A total of 169 samples from slaughter environment, raw beef at inspection and at public supply along Addis Ababa Abattoir Enterprise line were examined for Staphylococci . The isolates were tested against DNase, tube plasma coagulase, and eight medicinal drugs. A total proportion of 35.5% (60/169) isolates with 13.6% S. aureus and 21.9% coagulase negative Staphylococcus (CNS) were observed. All sampling locations were found positive for Staphylococcus environmental samples ranged from 18.2% - 46.2% with no difference (p > 0.05) among locations. In raw beef, it was 23.5% at abattoir and 52.9% at butchers. Three (1.9%), 13 (7.7%) and 23 (13.6%) of locations were positive for S. aureus only, CNS only and both as a mixed, respectively. Of all 60 Staphylococci isolates, the DNase test and coagulase tests were in agreement for 56 isolates (21 for positive, 35 for negative) showing DNase test was strong agreement with the gold standard test (coagulase tests), kappa=0.86)). S. aureus was 38.3% but CNS was 61.7%. Resistant isolates were observed for trimethoprim (35.0%), polymyxin-B (33.7%), oxytetracycline (31.7%), trimethoprim-sulfamethoxazole (20.0%), chloramphenicol (8.3%), oxacillin (6.7%), and gentamycin (5.0%) but not for tetracycline. Thirty-one (51.7%) isolates were resistant for at least one drug with multiple drugs resistance (MDR) of three to six in 17 isolates. Contamination of all sampling locations with Staphylococcus including with resistant isolate to medically used drugs warrants the application of good hygienic practices along the abattoir line. Due to availability and cost effectiveness, DNase can be used as alternative to the gold standard, coagulase test, for diagnosis of Staphylococcus . Keywords: Abattoir line; beef; drug resistance; foodborne intoxication; Staphylococci


Introduction
Staphylococcus species are classified as classical pathogen, causing infections (Waldvogel, 1995;Lowry, 1998). But, heterogeneous group of coagulase-negative Staphylococci (CNS) those historically as being less or nonpathogenic (Becker et al., 2014). They are differentiated based on their diagnostic reaction. Carriers are at higher risk of infection and they are presumed to be an important source of spread of S. aureus strains among individuals, to food and food production and processing chain (Muto et al., 2003;Kazakova et al., 2005;Miller and Diep, 2008). A fundamental biological property of S. aureus is the ability to asymptomatically colonize normal people. Approximately 30% of humans are asymptomatic nasal carriers of S. aureus (Kluytmans et al., 1997;Gorwitz et al., 2008). Due to patient and procedure related changes, CNS also represent one of typical opportunistic major nosocomial pathogens having a substantial impact on human life and health, and also food associated saprophytes resulted in clinically manifested infections (Becker et al., 2014).
The ideal identification of clinical S. aureus isolates requires a battery of tests and this is costly in resource limited settings. S. aureus identification was routinely done using either human or sheep plasma. Study was also conducted using mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings (Kateete et al., 2010). Indicating the absence of single phenotypic test (including tube coagulase) that can guarantee as reliable method for identification of S. aureus, Kateete et al. (2010) suggested the need for sequel testing of the isolates with Mannitol salt agar, DNase and Tube coagulase. On the other hand, the coagulase test is a golden standard method to differentiate S. aureus from coagulase negative Staphylococcus (Koneman et al., 1997). Coagulase production can be detected using either slide coagulase test or the tube coagulase test. The tube coagulase test is better than the slide coagulase test in that it detects the secreted extracellular free coagulase which reacts with a substance in plasma called "Coagulase-Reacting Factor" (CRF) to form a complex clot (Koneman et al., 1997;Winn et al., 2006).
The extended use and misuse of antibiotics in agriculture, livestock farming and human medicine have rapidly facilitated the emerging of resistant bacteria strains including Staphylococci (Valsangiacomo et al., 2000). Antibiotic resistance leads to prolonged hospital stay and increased costs in terms of treatment (Kitara et al., 2011). Multidrug resistance is norm among Staphylococci pathogens (Lowy, 2003).
Study along milk production, Gizaw (2014) reported Staphylococci species at 47.1% from udder milk, 58% from tank milk of the selected dairy farms, 34.4% from cow bucket swab, 38% from farm tank swab, 38% from milkers hand and 70% from nasal swab of milkers. Similarly, prevalence of 38.5% from carcass swab, 37.8% from knife swab, 48.6% from slaughter line swab, 37.8% from abattoir worker hand and 46.7% nasal swab of butchers were reported in Ethiopia (Gizaw, 2014). Indicating the significance of drug resistance, pooled prevalence of methicillin resistant S. aureus was reported at rate of 32.5% from several report meta-analysis data in Ethiopia (Eshetie et al., 2016). On the other hand, structured abattoir line-based Staphylococci survey with DNase test were not yet conducted in Ethiopia. Therefore, the aims of the study were to investigate beef abattoir line contamination with Staphylococcal strain, to evaluate DNase test as alternative of the tube coagulase test and to assess drug resistance status of the isolates against medicinal drugs frequently used in Ethiopia.

Abattoir lines description
The study was conducted at Addis Ababa Abattoir Enterprise (AAAE) located in Addis Ababa city. Multi-purpose cattle stocks purchased from extensive or semi-intensive management systems in different parts of the Ethiopia, transported to AAAE and slaughtered for human consumption were used. The AAAE has a capacity to slaughter up to 1,200 cattle in 8 hours with a staff of about 700 persons. After slaughter, carcasses were examined through routine meat inspection procedures. The raw beef, immediately or after a short cooling interval were loaded into meat transport trucks and delivered to city butcheries. Butcheries are mostly small open-stall shops, handling the meat at 20°C-27°C, which is the ambient temperature in Addis Ababa City. Hiko Ethiop. Vet. J., 2019, 23 (1), 90-110 Sampling and sample types Samples were taken from December 2011 to April 2012 from the abattoir and beef retailer butchery. In the abattoir, different samples were taken from the operation environment. Sample of raw beef were also taken directly after quality inspection at abattoir. Following the beef identification number, same raw beef product were sampled at butchery houses located at Addis Ababa. A total of 169 samples from 10 sampling locations (Table 1) were taken. For swabs, a 50 cm 2 area was swabbed with sterilized gauze moistened with normal saline solution. Water (20 mL) sample was filled directly from the tap into sterile calibrated glass bottles. Meat samples (34 samples from AAAE and 34 samples from butcheries) were taken aseptically and placed in sterile stomacher bags. The number and types of samples were described within Table 1 with the results. Samples were immediately transported to Microbiology Laboratory, Akililu Lemma Institute of Pathobiology, Addis Ababa University (ALIPB-AAU), Ethiopia on the day of sampling using an ice box at +4°C.

Sample preparation
Each sample was aseptically taken. For pre-enrichment, buffered peptone water (Merck, Darmstadt, Germany) was used and the inoculate were incubated at 37°C for 18-20 hours. The first 1:10 dilution was homogenized with a Stomacher 400 (Seward Laboratory, London, UK) and incubated at 37°C for 18-20 hours to be used as pre-enrichment ( Monttville et al., 2012;USDA, 2012).

Staphylococcus isolation and characterization
A loopfull of pre-enrichment was inoculated on whole-hen-egg based Kranep Agar (Merck, Germany). One to five presumptive Staphylococci colonies were taken per-each sample. Colonies were screened using 3% hydrogen peroxide. The presumptive positive S. aureus colonies isolate was transferred to Standard-II Nutrient Agar (Merck, Germany) incubated at 37 o C for 24 hr, stored at +4 o C. The strain was exposed to deoxyribonuclease (DNase) and tube plasma coagulase test in parallel. An overnight cultured colony was inoculated in forms of line ( Fig. 1) on DNase Agar (Merck, Germany) and incubated for 18-24 hr at 37 o C. The culture was over flushed with 1 ml 1 mole/ml Hydrochloric acid. Based up on DNA digestion zone of clear transparence surrounding the culture, the strains were termed as DNase positive. Parallel to DNase test, tube plasma coagulase test (Bactident Merck, Germany) was done. The colony was inoculated in Brain Heart Infusion (BHI) (Merck, Germany) incubated at 37 o C Hiko Ethiop. Vet. J., 2019, 23 (1), 90-110 for 18-24hr. A 0.5ml of rabbit plasma (Bactident Merck, Germany) was added in to sterile test tube followed by addition of 0.1ml of culture suspension. The mixture was incubated in 37 o C water bath with 1hr interval up to 4 hr with registration of degrees of coagulation. Finally, the tubes were kept overnight at room temperature and re-examined.

Considered and used test protocol
In Staphylococcus identification, coagulase production can be detected using either the slide coagulase test or the tube coagulase test (Winn et al., 2006). The tube coagulase is better than the slide coagulase test in that it detects the secreted extracellular free coagulase which reacts with a substance in plasma called "Coagulase-Reacting Factor" to form a complex clot (Koneman et al., 1997;Winn et al., 2006). Thus, coagulase testing was used as gold standard for differentia between S. aureus and coagulase negative Staphylococcus (CNS). Control reactions included templates of Staphylococcus aureus ATCC 25923 (positive control), Staphylococcus epidermidis ATCC 12228 and nuclease free water (negative controls).

Ethical Considerations
Samples were also collected from adult personnel's hands who are working in the abattoir. Individuals involved on the sample were informed regarding the details of the research objectives. Hence, informed consent was collected.

Antimicrobial resistance test
The test was done on Mueller-Hinton agar (Oxoid, Hampshire, England) using Kirby Bauer disc diffusion criteria of the National Committee for Clinical Laboratory Standards (Bauer et al., 1996;CLSI, 2012). The following antimicrobial substances (Oxoid UK) were used: chloramphenicol (C 50 µg), gentamycin (CN 10 µg), oxytetracycline (OT 30 µg), oxacillin (OX 1 µg), polymyxin B (PB 300U), tetracycline (TE 10 µg), trimethoprim-sulfamethoxazole (STX 1.25/23.75 µg) and trimethoprim (W 5 µg (Oxoid, Hampshire, England). They were selected using the fact that they are used in health sectors of Ethiopia and their availability on the markets. The isolates were sub-cultured on Nutrient agar (Merck, Germany) and incubated at 37 o C for 24 hrs. They were then inoculated into 5 ml of Brain Heart Infusion broth (BHI) (Merck, Germany) and again incubated for 1 hr at 37 o C. The inoculum density was standardized using a 0.5 McFarland standard. Volume of 0.1 ml of standardized culture was spread on Mueller-Hinton agar (Oxoid, Hampshire, England). The antibiotics disc was mounted and incubated at 37°C for 18-20 hours. Based on the diameter of inhibition zone, the results were recorded as susceptible, intermediate and resistant according to the rules of National Committee for Clinical Institute laboratory standards (CLSI, 2012).

Data analysis
Data were entered in to Microsoft Excel 2007® (Microsoft Corp., Redmond, USA) and analyzed using Excel, State 11, and SPSS version 20 (IBM Corp., Armonk, USA). Percentage and 95% confidence intervals (CI) of the percentage were used to demonstrate prevalence differences between and among the sampling occasions and types of samples. Using the coagulase test as gold standard (Winn et al., 2006;Koneman et al., 1997), the diagnostic test agreement (Kappa), sensitivity and specificity were also calculated. The cut-off Kappa value and interpretation was according to Viera, and Garrett (2005).

Results
Out of the total 169 samples, 35.5%; 95% CI 28.6%-42.9% were found positive for Staphylococcus. The prevalence ranges of 18.2% in hooks to 46.2% from personal hand swab with no difference (p > 0.05) were observed in environmental samples. In raw beef, it was 23.5% from when ready for distribution at abattoir and 52.9% in post distribution at butchers (Table 1). From a total sample, 13.6% S. aureus and 21.9% CNS were observed (Table 2). Among environmental samples, S. aureus ranged from 0% on knives to 36.4% in meat transport trucks while CNS ranged from 0% on hooks to 46.2% on personnel's hands swabs. In raw beef, S. aureus was 8.8% at ready for distribution but raised to 20.6% post-delivery at public supply. Similarly, CNS was 14.7% at ready for distribution but raised to 32.4% post-delivery at public supply.   The coagulase test was used as gold standard. Of all 60 isolates exposed to DNase ( Figure 1) and coagulase tests, 21 were positive for both tests whereas 35 were negative for both test reactions showing (Kappa = 0.86; 95% CI = 0.73-0.99) Sensitivity = 91.3% and Specificity = 94.6%) ( Table 4). Out of the total 60 isolates, 23 (38.3%) and 37 (61.7%) isolates were coagulase positive Staphylococcus (CPS) and coagulase negative Staphylococcus (CNS), respectively.

Antimicrobial Resistance test
The 60 Staphylococcus isolates were subjected to 8 types of antibiotics for antimicrobial resistance test. Out of the total isolates, 35.0% were resistance to trimethoprim, 33.7% for polymyxin B, 31.7 % for oxytetracycline, 20.0% for trimethoprim-sulfamethoxazole, 8.3% for chloramphenicol, 6.7% for oxacillin, 5.0% for gentamycin and no resistance was observed for tetracycline (Table 5).

Antimicrobial resistance at different sampling sites
Two (5.9%) to 12 (35.3%) resistant Staphylococcus isolates from abattoir environmental samples was observed against all drugs used in this study. Similarly, resistant isolates to drugs used in this study were distributed on beef at abattoir and butchery. Exceptions are oxacillin resistant isolates were not observed on beef from abattoir and butchery while gentamycin resistant isolates were not observed in beef at abattoir (Table 6).   (Table 7). Two (15.4%) to ten (76.9%) resistant S. aureus isolates to chloramphenicol, gentamycin, oxytetracycline, oxacillin, polymyxin B, trimethoprimsulfamethoxazole and trimethoprim were observed at abattoir environment (Table 7). On the other hand, resistant Staphylococcus isolates to chloramphenicol, gentamycin and oxacillin was not observed on beef both at abattoir and butchery. However, regardless of the number, high percentages of resistant Staphylococcus isolates to oxytetracycline, polymyxin B, trimethoprimsulfamethoxazole and trimethoprim were observed on beef at butchery than at abattoir. With regards to CNS, variable types of resistant isolates to the used drugs were observed at each sample origin.

Resistance pattern
Out of the tested 60 isolates, 31 (51.7%) were resistant to one drug to multiple of six drugs (Table 8). Ten isolates for single drug, four for two drugs but the remaining 17 for multiple of three to six drugs resistance (MDR) were observed. Higher isolates of 5 were found resistant to four dug combination of oxytetracycline-polymyxin B-trimethoprim-sulfamethoxazole-trimethoprim (OT-PB-SXT-W).

Prevalence of Staphylococcus
Meat is an important vehicle for the transfer of antibiotic resistances from animals to humans, and antimicrobial resistance has always been a major concern for nosocomial infections in hospital environments. Such transfer can occur in three ways: by means of antibiotic residues in food, through the transfer of resistant food borne pathogens, or through the ingestion of resistant parts Hiko Ethiop. Vet. J., 2019, 23 (1), 90-110 of the original food microflora and resistance transfer to pathogenic microorganisms (Kruse and Sorum, 1994;Klein, 1999;Teuber, 1999;Mayrhofer et al., 2004). The present overall prevalent of 35.5% of Staphylococcus is slightly lower than the 42.8% total Staphylococcal reported by Gizaw (2014). However, there is similarity in prevalence of S. aureus at hooks (18.2%) and personnel hand swab (46.2%). The raising Staphylococcal load from 23.5% at ready for distribution at abattoir to 52.9% butchers house indicated the high risk of beef contamination by S. aureus carriers human being and from the beef handling environment like transporting trucks (45.5%) along the meat value chain (Gudeta, 2012). Asymptomatic nasal carriers of S. aureus (Kluytmans et al., 1997;Gorwitz et al., 2008;Gizaw, 2014) as a normal flora with human an important source of spread of S. aureus to food and food production and processing chain were also indicated by Miller and Diep (2008), Kazakova et al. (2005) and Muto et al. (2003). This was supported by 46.2% Staphylococcal isolation on personnel's hands swabs observed from the current study.
The present findings of S. aureus (8.8%) and CNS (14.7%) were lower than the S. aureus prevalence 38.5% from carcass swab (Gizaw, 2014), 28.57% from poultry and 29.41% from beef, 23.86% S. aureus from raw meat (Pesavento et al., 2007) but similar was 15.15% from pork (Pesavento et al., 2007). But the prevalence on ready for distribution and the 20.6% S. aureus and 32.4% CNS were similar with reports of Gizaw (2014). This variation could due to difference in study area and sources of sample. Moreover, the present concomitant raising of S. aureus (8.8%) and CNS (14.7%) at ready for distribution to raise to 20.6% and 32.4% respectively at post-delivery at public supply could be due to further contamination of the beef product at postharvest stages followed by growth of microbial. These indicate the increasing in rate of contamination and multiplication of Staphylococcal on the carcasses and along the beef handling stage. Rani et al. (2017) suggested meat distribution stage as the most critical period where the quality can easily be compromised. During distribution, pathogenic and spoilage micro-organisms may grow (Rani et al., 2017).

DNase and Coagulase test
Coagulase testing was used as gold standard for differentia between S. aureus and coagulase negative Staphylococcus (CNS) (Winn et al., 2006). Clinical S. aureus isolation requires a cost effective and better tests in resource limited developing countries (Kateete et al., 2010) where tube plasma coagulase test is confirmatory. However, as an alternative diagnostic tool, DNase test can assist Hiko Ethiop. Vet. J., 2019, 23 (1), 90-110 or replace it. The present finding showed almost perfect agreement (Kappa = 0.86, 95%CI = 0.73-0.99) between the DNase and tube plasma coagulase tests in screening S. aureus from the CNS. According to Viera and Garrett (2005), almost perfect test agreement ranges from 0.81-0.99.
The present 91.3% sensitivity was found in agreement and lower than 94-100% previous findings reported by other investigators (Tager et al., 1948, Orth et al., 1971Oranusi and Umoh, 2006)

Drug resistance profile of Staphylococcal species
The rapidly increasing in the number of antimicrobial resistant bacteria agents could be due to the extended use and misuse of antibiotics in livestock farming and human diseases treatment). Staphylococcal species (Kitai et al., 2005;Becker et al., 2014), have shown a considerable increase in resistance against most antibiotics (Valsangiacomo et al., 2000) with the consequence of prolonged hospital stay and increased costs of treatment (Kitara et al., 2011). Tetracycline resistant isolate was not observed in this study but 62.5% were reported from milk (Gizaw, 2014). Again, unlike the present finding, 19.0% S. aureus were resistant to tetracycline with 8.3%, 20.0% and 25.0% from poultry, beef and pork were reported (Pesavento et al., 2007). This could be due to the difference among the sources of isolates and geographical location. On the other hand, the 31.7% resistant isolate to oxytetracycline observed in this study could be from frequently use of this in Ethiopia in animal's treatment (DACA, 2009) which might develop drug resistance. Present finding showed higher resistant S. aureus (43.5%) than CNS (5.4%) to co-trimoxazole indicating greater risk of resistance development of the pathogenic strain than the less pathogenic group to commonly used drug. The current 20.0% resistance to co-trimoxazole was lower than the resistant S. aureus from outpatients (53.6%) and inpatients (44.7%) (Kitara et al., 2011). The overall 6.7% oxacillin resistant Staphylococci with S. aureus (13.1%) and CNS (2.7%) were lower than the 35.7% resistant S. aureus isolates (Pesavento et al., 2007). This finding is still lower than the 66.7% from poultry and 30.0% from pork oxacillin resistant S. aureus. But the present oxacillin resistant S. aureus was similar with the 10.0% reports from beef meat items (Pesavento et al., 2007). The current over all 5.0% resistance to gentamycin was similar with a pooled 9.5% resistant S. aureus reported by Pesavento et al. (2007) but lower than the 16.7%, 10.0% and 5.0% from poultry, beef and pork (Pesavento et al. (2007) respectively.
The present 15.4% to 76.9% resistant S. aureus to chloramphenicol, gentamycin, oxytetracycline, oxacillin, polymyxin B, trimethoprim-sulfamethoxazole and trimethoprim were observed at abattoir environment could be due to contamination of the abattoir environment from animals, or from carrier humans particularly from abattoir workers harboring resistant strain. Evidence that transfer of antimicrobial resistance from food-producing animals to humans directly via the food chain as route of spread were documented (Wooldridge, 2012). The absence of resistant Staphylococcus isolates to chloramphenicol, gentamycin and oxacillin on beef both at abattoir and butchery could be due to the fact that these drugs are not used in animal treatment in Ethiopia where no development of resistant strain. The occurrence of one or more resistant almost along the studied beef line location indicates the contamination of environmental sites which concerned as one routes of resistant isolate circulation via contact. Contaminated environmental sites were suggested as pathway for the transfer of resistant isolate in meat (Wooldridge, 2012;Bengtsson-Palme et al., 2018). Moreover, transfer of resistant genetic elements between bacteria in mixed populations as a cross resistance to drugs of similar generic groups could make complex potential routes of resistant isolate spread. Similar suggestions were given (Wooldridge, 2012;Bengtsson-Palme et al., 2018).
Total of 51.7% resistant isolates to one drug to multiple of six drugs consisting as multiple drugs resistance (MDR) were observed. A large proportion (89.3%) from food line (Gizaw, 2014), 53.7% from hospital cases (Gizachew et al., 2015) were showed MDR in Ethiopia. The presences of nasal carriage of resistance staphylococcus were also reported by Tewodros and Gedebou (1984) in Ethiopia. Like the present 51.7% MDR isolation, Pesavento et al. (2007) also reported 30.95% resistant isolates to at least three antibiotics in indicating significance resistance among these pathogens.

Conclusion and recommendation
The present finding indicates increased contamination of meat product along handling line with both by S. aureus and CNS with more than half percent of the isolates being resistant to single to multiple drug resistant. Thus, this finding warrants the application of good hygienic practices along the beef abattoir line with control of drug resistance. The DNase test showed strong agreement with tube coagulase test with low false positive and negative isolates indicated the possible alternative use of these diagnostic tests for screening staphylococcal isolates in resource-limited settings like Ethiopia.