Sys Rev Pharm 2020;11(11):464-475

A multifaceted review journal in the field of pharmacy Characterization of Foodborne Pathogens and Enterotoxigenic Isolates with Detection of Antibiotic Resistance from Beef

Suad A Al-Hilua*, Wisam H Al-Shujairib

a bCDoerpraerstmpoendt ionfgBiaoulothgoy/rF: aculty of Science, University of Kufa, 54001, Najaf, Iraq *Department of Clinical Laboratory Sciences/College of Pharmacy, University of Babylon, 51001, Hilla, Iraq [email protected] Abstract This study aimed to determine the current status of Keywords. , Meat products, Foodborne bacterial contamination in the animal products provided , Bacterial counting and to analyze isolates, Staphylococcus Corresponding author: [email protected] aureus, and from samples. The status of bacterial contamination was investigated in a total of 52 samples of beef meat intended for human consumption purchased from a general meat shop from September 2019 to December 2019 in Iraq. All meat samples that collected from butchers were contaminated by microorganisms. Isolation and characterization of the bacterial specimens from the samples were performed using conventional cultural techniques and biochemical identification. Five species of bacteria were isolated from all samples including Salmonella, E. coli, Staphylococcus aureus, Shigella, and Enterobacter faecalis. Three species of bacteria showed significant contamination of meat, these are Salmonella, E. coli, and Staphylococcus aureus. Salmonella were recovered from beef meat 30(58%), while E. coli 15(29%) and Staphylococcus aureus 20(38%). Antimicrobial susceptibility against ten antimicrobial agents commonly used in human was tested by used the disc diffusion method, including Ampicillin (A)(10µg), Amoxicillin/clavulanic acid (AM)(10 µg), Chloramphenicol (C)(30 µg), Streptomycin (S)(10 µg), Trimethoprim (Tr)(5 µg), Tetracycline (T)(30 µg), Ciprofloxacin (Cf)(5 µg), Nalidixic acid (Na)(30 µg), Gentamicin (G)(10 µg), and Kanamycin (K)(30 µg). multidrug resistance was detected in most of bacterial isolates, all isolates of Salmonella were resistant to streptomycin, amoxicillin, chloramphenicol, and gentamicin but sensitive to other antibiotics. PCR assay for detection of enterotoxin gene (sea gene) of S. aureus isolates, the result revealed that only 15 isolates from 20 isolated S. aureus carried this

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Vol 11, Issue 11, Nov-Dec 2020 Sys Rev Pharm 2020;11(11):464-475

A multifaceted review journal in the field of pharmacy gene and showed bands after electrophoresis examination. We conclude, the existence of foodborne pathogens and indicator organisms such as toxigenic Staphylococcus aureus in meat shows bad food handling and processing practices. Consequently, butchers and meat suppliers should be educated about the side effects of lack of adequate personal and environmental and sanitation. In addition, consumers must avoid consumption raw and undercooked foods. These results suggest that meat can be a source of resistant bacteria, which can spread to society through the food chain. We conclude that resistant strains of bacteria in beef meat are common. These results support the adoption of strategies for the wise use of antibiotics in food animals and for reducing the number of pathogens existing on farms and slaughterhouses.

INTRODUCTION E. coli

reservoir for Enterobacteriaceae especially where Food safety is an important concern that has increased in contamination occurs during the process of slaughter and international trade. Outbreaks of food-borne pathogens sacrifice of the carcass remains unhealthy and the are one of the important things that leads to illness and proliferation of large number of bacteriSaalmgeonneerlalates a death, about 24-81 million of food borne disease public health hazard [14]. Contaminated meat can able to associated with meat every year were recorded [1]. The transmitted of zoonotic infections [15]. are eating of contamination foods with pathogenic bacteria the main cauEs.actiovlei agents of foodborne infections in and their products such as and enzymes leading to human and resulted outbreaks in USA and serious diseases [2]. Food spoilage mean any change in Europe [16]. is indicated to the presence of fecal food such as taste, smell, and appearance, but it remains contamination in food directly or indirectly. The safe for the consumer until the number of conventional methods for detection of pathEog. ecnoliic microorganisms present in the food reaches a certain Sbalcmteorniaelilna fSohoidgeallrae pPerrofteecutsby which the selective media limit that causes food-borne diseases [3]. Meat and meat were used for identification of morphology [17]. , products are considered a fertile environment for , , , and others are geSnaulms onf etlhlae EmsicchreoroircghaianicsomlisSatnapdhtyhloeciroctcouxsinasu,rienucsluSdtirnegptboaccotcecruisa [4]. efanmteirlyicaEnterToybpahcitmeruiraicuemae and its presence in meat SOhniegeollfathe moSsatlmimopnoelrltaant bacteriaCliossotlraidteiudmfropmerfmrinegaetnis indicates to enteric contamination [6]. , , spp., and are the main strains associated spp., spp., and with human salmonella infection (salmonellosis). The [5]. Food-borne pathogens are colonize the contamination of meat products with these strains are gastrointestinal tracts of consumed domestic animals by the main source of infection [18]. Therefore, consumption human [6]. The slaughtered animals are sterile but of uncooked beef can pose severe health complications despite this, meat gets easily contaminated with for consumers [19]. Salmonellosis leftovers one of the microorganisms by several processes [7]. Most of these most foodboSranlemondeislleaases worldwide, especially in organisms transmitted into meat and meat products developing countries. The appearance of antibacterial through the hands and clothes of workers and resistance in isolates from food products can contaminated environment, devices, and knives [8]. Food- influence the treatment of this infection [20]. borne microorganisms are a major source of disease and In developing countries, the food-borne diseases not death, leading to significant spending on health care. recognized and not reported. Therefore, the statistical Children are more susceptible to food poisoning because data are continuous increased [21]. In fact, even cleaning of their weak immune system [9]. There are two types of and disinfection process of product surfaces to reduce the microbial contaminants, microorganisms that capable to adherent bacteria, but it does not remove all bacteria, and produce disease and the second type that spoils the meat a small amount of them remains on surfaces and devices, products and makes them unfit for human consumption and therefore it can be transmitted directly to meat by [10] .The increase consumption of meat especially the knives, slicers, or conveyor belts or indirectly by floors contaminated food borne bacteria because including [22]. The emergence of antibiotics and their excessive proteins, vitamins, lipids, minerals and other nutrients uses have generated resistant strains of bacteria. In resulted outbreak of food-borne infections [11], possibly animals, antibiotics have been used to stimulate growth due to the presence of large amounts of water in meat, it and cure diseases. This in turn has led to the generation provides an environment suitable for bacterial growth of resistant bacterial strains that are transmitted to [12]. Meat contains about 75% of water, 19% of protein, humans through eating their meat [23]. 2.5% of fat, 1.2% of carbohydrates, and exist of vitamins, The present study aimed to highlight on the prevalence minerals , and cholesterol [13]. Cattles are the main and enumerate of foodborne pathogens from beef meat, it 465 Systematic Reviews in Pharmacy

Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat

ᵒ was bought from different butcher shops. Isolation, inoculated and streaked onto MacConkey agar. The plates identification, and determine the antimicrobial resistance Swaelmreoinneclulabated at 37 C for 24-48 hours and then it picks of the bacterial pathogens from beef meet. Also, verify up colorless and transparent colonies supposed to be MstaApThEyRloIcAoLcScaAlNvDiruMleEnTcHe OgeDnSes. and is subject to subsequent processing Place Selection: including motility test, Gram's staining, and biochemical tests. To obtain pure cultureᵒ the colonies from Places for taking meat samples were MacConkey agar can be subcultured on Salmonella chosen from butchers that are characterized by failure to Sahlimgeollnael(lSaS) agar, incubated at 37 C for 24-48 hours. The Soabmseprvle tChoellheecatilothn:conditions to maintain human health colorless colonies with black center were regarded as and safety from diseases. Shigella anIdsoslautbijoencteodf Sttoapfhuyrtlohceorccsutusdayu, rweuhsil.e the A total of 52 samples of beef meat colorless colonies that grew on SS agar are considered as were collected from different places in Al-Najaf Al-Ashraf -1 [27]. 1 ml Province/Iraq. These places included Al-Krama, Al-Naser, of 10 dilution from meat sample wᵒ as inoculated and Al-Jazeera, Al-Hindia, Al-Askari, Al-Wafaa, Al- streaked onto Mannitol Salt Agar (MSA) as a selective anSd. Muhandiseen, Al-Nafut, Al-Ansaar, Al-Jamiaa, Al-Jameia, aduifrfeuresntial medium, incubated at 37 C for 24-48 hours. Al-Nedaa, and Al-Melaad Quarter. Samples collected were Yellow colonies with yellow zones pSi.ckauupreauss positive purchased from butchers to the consumer. From each Staphy,lowcohcicleusceoploidrelersmsidainsd red colonies with red zones area, four samples were collected, and each sample Gwrearme 'sstsatpahinyilnogcoMcceithootdh:er than such as weighs 250 grams. After collection all samples, they are [28]. brought transferred in sterile plastic bags directly to the one drop of water is placed on Laboratory of Microbiology at the Faculty of Science, slide and a small colony of potential colonies cultured on SUanmivperlesitPyroefpKaruafati,oan:d placed in clean containers in order Brain Heart Infusion agar is taken and placed over a drop to isolate and diagnose the bacteria in it. of water and mixed well until it 2becomes white and From each sample, about 1 gram of spread on slide with an area of 1cm . The smear is fixed meat is cut with a clean, sterile knife, then the surface by air or gentle heat. First, crystal violet was applied on SlaaymerpelexpPorsoedcetsosianirg:is removed, the samples are weighed the smear for two minutes and washed with running and prepared for subsequent processing. water. Second, some drops of iodine were added for one Each sample of meat is subject to minute and washed with tap water. Third, add acetone two ways to isolate the bacteria, including: The first alcohol for few second and washed with tap water. Forth, method, the sample is taken and placed directly on a add safranin for two minutes as a counter stain and brain heart infusion agar medium. Bacteria grow around washed with water. The slide leave for dried in air and and under the meat sample. The second method is the BexiaomchineemdicuanlderTtehsetsm: icroscope with power 40x and then meat cut into small pieces and placed in a buffer to the 100x by using oil immersion [29]. second day, then 1-2 drops of solution are taken and All bacterial isolates were Bcualctuterreidal oCnoubnrtaiing:heart infusion agar medium by biochemically tested by using IMViC pattern including streaking method. indole (I), methyl red (M), voges-proskauer (Vi), and All samples were subjected to citrate utilization test (C). addition to that Kligler's iron bacterial counting by taking 25 gram of meat sample, they Satgaaprh(yKloIcAo)c,curseasueretuesst, and oxidase test also used for are added to 225 ml of buffered peptone water (BPW) in identification of bacteria. For the diagnosis 500 ml Erlenmeyer flask, leave for 1-2 min for Methyl red test: , coagulase and catalase tests are homogenized and make serial dilutions out of them. used. Serial dilutions were done with ten test tubes containing Inoculate the liquᵒid medium of glucose 9 ml of no-r1mal-10saline or phosphate buffer solution and phosphate peptone water from a young agar culture of labeled 10 -10 . Add 1 ml-1of diluted meat sample into the test bacteria and incubate at 37 C for 48 hours. Add 5 first test tube to formed 10 dilution, then transferred 1 drops of the methyl red reagent. Mix and read ml of fir-2st tube to the second tube after well mixed -t1o0 Vimomgeesd-iParteolsyk. aPuoesrititveestt:ests are red color and negative form 10 dilution and so it continues un-4til re-a5ch to 10 -6 tests are yellow color [30]. dilution. 1-2 drops of ᵒsample from 10 , 10 , and 10 ᵒ Inoculate single colony of test dilutions were inoculated on triplicate nutrient agar bacteria in glucose phosphate peptone water. Incubate at plates, incubated at 37 C for 24-48 hours. The bacterial 37 C for 48 hours, then add 1 ml of 40% potassium DcoeltoenciteisowneorfeBcaclctuelraitaeldSwpeitchieCso:loIsnoyl-aFtoiormn ionfgEU.nciotl(i.CFU) hydroxide and 3 ml of 5%solution of α-naphthol in [24, 25]. E. coli Cetihtraantoel.uAtifltiezrat5ionmtinests:howed pink color indicated to The positive reaction [30]. ability of to ferment lactose gives us an option to ᵒ Inoculate a small colony of the choose the MacConkey agaSralmfoornedlilsacriminSahtiegeblleatween test organism onto Simmon᾽s citrate medium. Incubate lactose fermenting strains from other lactose non- for 96 hours at 37 C and read the results. Positive result fermenting-1strains, such as and . Take Krelpigrleesre᾽nsteIdrobnluAegcaorl:or with growth, negative result is 1 ml of 10 dilution and inoculated onto MacᵒConkey agar original green color and no growth [30]. medium and cultured by spreading method by using glass Slants of kligleᵒ r iron agar were spreader. Incubated the Petri dishes at 37 C for 24-48 inoculated with stock culture of bacteria in the hours. TheE.ccoolloinies which characterizedE. bcyolired/pink Ucornevaesnetitoensatl: method. Incubated at 37 C for 24 hours and color, round, medium sized, and non-mucoid picked as read the result [31]. suspected colonies [26]. Expected colonies Sterilize basal medium without glucose or can be confirmed by gramIssotlaaitnioanndofbSioaclhmeomnieclalla taenstds urea (which sterilize by filtration). Cool to 50and add the Swhhiigcehllian.cluding indole test, methyl red test, oxidase test, glucose or urea to reach final concentration of 2%. and voges-proskauer -t1est. Dispense into tubes as deep slopes and inoculate with 1 ml of 10 dilution from meat sample was heavy growth of bacteria on the surface of the slants for 4 466 Systematic Reviews in Pharmacy

Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat

Odaxyids aseincteusbta:tion. Positive result represented by The results of resistance, intermediate, and sensitive development of a purple-pink color [32]. were performed according to criteria of National This method described by Kovacs [33], a DCoNmAmEitxttereacftoiornC: linical Laboratory Standards (NCCLS, small piece of filter paper was soak in 1% of Kovacs 2004) [37]. oxidase reagent and by a loop pick a colony from fresh Phenol/Chloroform method was used bacteria and spread on the filter paper. Wait and for extraction of ᵒgenomic DNA according to Janet and observed the change in the color, purple color indicates Adel (2006) [38]. The bacterial isolates were cultured on Cthaetaploassietivteesotxidase test. While the color does not change LB medium at 37 C for 24 hours. Centrifuged the grown mean the microorganisms are oxidase negative. isolates at 6000rpm for 2min, discard the supernatant : A colony of bacteria was taking with a and the pellet was suspended in 400 µL of STE buffer toothpick and mixed with a one drop of hydrogen which consist ofᵒ 2%SDS, 100mM NaCl, 100mM Tris-HCl Cpeoraogxuidlaeseonteast:slide. Positive result by indicates the and 10mM EDTA with pH=8.0, after that the solution was effervescence [9]. incubated at 55 C for 30min. Add 200 µL of phenol and 0.2 ml of overnight broth bacterial 200 µL of chloroform to the solution and centrifuged for culture was addᵒed to 0.5 ml of diluted rabbit plasma 10min at 8000rpm, then transferred the upper aqueous (plasma: saline 1:5) in a tube. Gentle mixing the tube and phase to a clean tube and adding 100 µL of ice cold Dinectuebcattieodn aot f37AnCtifboirot2i,c4,Su2s4cehpotuirbsi.liPtyo:sitive result by isopropanol for precipitated DNA. Centrifuged for 10min observed the clot in the tube [34]. Pato1ly0m00e0rrapsme , finCahllayi,nthe pReelalecttiwonas disAssoslavye:d inP5r0imµLerosf An antibiotic PTErebpuafrfeart.ion. sensitivity test (AST) is usually performed to determine which antibiotic is most effective in treating bacterial The primers were prepared in a lyophilized infᵒection in an organism. Add 1-2 colonies of each form by which they dissolved in TE buffer to give final bacterial samples to 5 ml of nutrient broth, incubated at concentration of 100pmol/µL. From the stock solution 37 C for 24 hours and then mixing and homogenized very Sta. kaeur9eµuLs and mixed with the 100 µL oeftTaEl.buffer, this is well. 1 ml of bacterial suspension was inoculated on the working solution. The primer of enterotoxin gene for plates containing Mueller-Hinton Agar (MHA), spread the was obtained from Sharma (2000) [39], suspension over all the agar surface even the edges by the sequencPeCsR Mixotfure. this primer (F: sterile glass spreader. Inserted the cultured plates into TGTATGTATGGAGGTGTAAC, R: ATTAACCGAAGGTTCTGT) the fridge for 30 min to absorb the bacterial suspension at 270 bp size. The final total volume of before putting the antibiotic discs [35]. The antibiotic PCR mixture in 20 ul included 5 ul of DNA samples, discs that seeded onto the agar included Ampicillin Forward primer 1.5 ul, Reverse primer 1.5 ul, and sterile (A)(10µg), Amoxicillin/clavulanic acid (AM)(10 µg), deionized water 20 ul in Eppendorf tube containing PCR Chloramphenicol (C)(30 µg), Streptomycin (S)(10 µg), Premix. Trimethoprim (Tr)(5 µg), Tetracycline (T)(30 µg), PCR Cycling condition: PCR tubes were placed into Ciprofloxacin (Cf)(5 µg), Nalidixic acid (Na)(30 µg), thermocycler PCR instrument where DNA was amplified TGeanbtlaem1i.cin (G)(10 µg), and Kanamycin (K)(30 µg) [36]. S. auraesuisndicating in below (Tab.1). Stage Temperature (time) Program used for amplifying the enterotoxin gene oᵒf ᵒ Initial denaturation 94ᵒC for 30 s Denaturation 92ᵒC for 30 s 52 cycles Annealing 50ᵒC for 30 s Extension 72 C for 30 s Final extension 72 C for 2 min

Figure 1.

Comparison between two methods for isolation of bacteria from meat samples, to left, 1g of meat sample put directly on culture medium, to right, I drop of buffer solution containing meat sample streaked on culture medium.

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RESULTS Figure 2.

Light microscopic view of Enterobacter faecalis In this study, two methods were used to isolate bacteria from meat samples (Fig.1), which is the method of placing 1g of meat sample directly on brain heart infusion agar and the method of placing the meat sample in a buffer solution, then taking drops of buffer and streaking on brain heart infusion agar. The second method was the best because of the isolation of many types of bacteria compared to the first method. The results showed all meat samples that collected from butchers were contaminated by microorganisms. The samples which collected from most butchers especially from north quarters gave a high levelSoaflmcoonntealmlainatioEn. .coli A tSotapl hoyfloc5o2ccuissoalautreesu, sfive Sshpigeeclileas of baEcnteterriaobwacetreer fisaoelcaatleisd from all samples including 30, 15, 20, 6, and Salmonella Staphyloc3occ(uFsiga.2u)r.euTshree Es.pceocliies of bacteria showed Tsiagbnilfeic2a.nt contamination of meat, these are , Salmonella , and (Tab. 2). S. aureus E. coli IsColalaritfeydthBeacdtiestrriiabuRteiognioonf bacterial isolates aSccaolmrdoingeltloathe places taken froEm. ctohleim, where we nSotaeptha. taureus is the most frequently observed in Al-Jazeera and Al-Anaasr quarter followed by and then .

Al-Krama 1 1 2 Al-Naser 2 1 1 Al-Jazeera 4 2 3 Al-Hindia 3 1 1 Al-Askari 3 1 1 Al-Wafaa 1 1 1 Al-Muhandiseen 1 1 2 Al-Nafut 2 1 2 Al-Ansaar 4 1 1 Al-Jamiaa 1 1 1 Al-Jameia 3 2 1 Al-Nedaa 3 1 3 Al-Melaad 2 1 1 Total Salmonella 30(58%) 15(29%) 20(38%) Staphylococcus aureus E. coli Salmonella The predominant species was (58%), followed on SS agar (Fig. 3), examination with gram stain, and by (38%), then (29%). Out biochemical tests. colonies will appear of fifty-two meat samples coSllaelcmteodnelflraom thirteen colorless with black centers because these bacteria do not butchers exist in different places in al-Najaf only 30 ferment lactose, they are a product of hydrogen sulfide samples were contaminated with by culturing gas.

Figure 3. Salmonella Shigella Two species of bacteria streaking on Salmonella-Shigella agar. Colorless colonies with black center of on the left, yellow colonies of on the right. Salmonella The light microscopic examination of presumptive cells quite large about 0.7-1.5 µm in diameters, bacillus to isolates, the results showed Gram-negative rod shaped (Fig. 4).

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Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat

Figure 4.

Salmonella species unSdaelmr tohneelliaght microscope, showed gram negative cells bacillus to rod shape.

While, the results of biochemical tests of simmon᾽s citrate (+), urease (-), kligler᾽s iron agar 2 showed indole (-), methyl red (+), voges proskauer (-), (Alkaline/Acid with H S), and oxidase (-) (Fig. 5) (Tab. 3).

Figure 5. Salmonella

Biochemical tests for spp. show positive results in a test simmon᾽s citrate (right) and kligler᾽s iron agar, Table 3. alkaline/acid with H2S. Biochemical Tests Bacterial Biochemical testMs eotfhthyrlee specieVs ogf ebsa-cteria isolatSeidmfmroomnmeat species Indole urease KIA Oxidase Coagulase Catalase red proskauer citrate Salmonella E. coli S. aureus - + - + - K/A - nil nil + + - - - A/A - - + nil nil nil nil nil nil nil + + Staphylococcus aureus S. aureus A total of 52 meat samples were used in this study, only like cluster. ᵒ 20 (38%) samples were positive to S. All strains of were cultivated on 5% of sheep abuyrecuslturing on mannitol salt agar, the golden yellow blooSd. aaguarre,uisncubated at 37 C for 24 h, then identified by colonies with smooth round represented presumptive biochemical tests (Fig. 6). The result of biochemical tests bacteria. On the other hand, the microscopic for revealed positive for both coagulase and examination showed gram positive bacteria with grape catalase.

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Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat

Figure 6. S. aureus

The figure referred to the ability of to hemolysis of blood agar, where it was found that the strains non S. aureus hemolysin for blood. 3 The count number of colonies are 4.98×10 agar as selectiEv.ecoalnid differential medium that used for CFU/g in Al-Na3ser QSutaarptheyr,lotchoisccruessult show lowest count, isolation and differentiation of Enterobacteriaceae (Fig. Ew.hcioleli 6.45×10 CFU/g in Al-Nedaa Quarter represented 7). Colony of on MacConkey agar showed red or the highest count of . pink color (because the production of acid from lactose) was confirmed in 15 isolates from 52 meat samples and non-mucoid. through morphological characteristics on MacConkey

Figure 7.

Pink color and non-mucoid colonies of E. coli because the production of acid from lactose, cultured on MacConkey E. coli agar plate. 3 Mean counts of were 2.26×10 CFU/g fo3r local for small butcher's shops in Al-Nedaa Quarter (Fig. 8). butcher's shops in Al-Naser Quarter and 4.35×10 CFU/g

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Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat

Figure 8. E. coli E. coli

Counting of isolates isolated from meat samples, two Petri dishes cultured by one drop of serial dilutions.

The isolated pathogens were evaluated for susceptibility chloramphenicol, streptomycin, trimethoprim, to 10 antimicrobial agents used in humans. Resistance of Steatlrmaocyncellilnae, gentamicin, and kanamycin. Multidrug bacterial isolates to antimicrobials was commonly resistance was observed in three isolates. All isolates of observed. The majority of these bacteria revealed high were resistant to streptomycin, amoxicillin, resistance to several antimicrobials, especially ampicillin chloramphenicol, and gentamicin but sensitive to other Tanadblgee4n.tamicin (Tab. 4). bacterial isolates were resistant antibiotics. to Bacatmerpiiaclillin, amoxicillin/clavulanic acid, Antibiotics IsolNautemsber, percenAtage of isoAlaMtes resistanCt to antibioticSs (%) Tr T Cf Na G K Salmonella S. aureus E. coli n=30 30(100) 30(100) 25(83.33) 10(33.33) 0 0 0 0 30(100) 0 n=20 10(50) 10(50) 0 0 20(100) 16(80) 0 0 19(95) 0 n=15 15(100) 15(100) 0 S. 0 0 1(6.66) 0 0 0 5(33.33) aureus sea S. aureus An antibiotic susceptibility test was performed on all trimethoprim, Ciprofloxacin, nalidixic acid. isolates for 10 antibiotics; resistance recorded in The detection of enterotoxin gene ( gene) of S. 100% E.focorli ampicillin, amoxicillin, trimethoprim, aisuorleautess by using conventional PCR assay (Fig. 9), the tetracycline, and gentamycin. result revealed that only 15 isolates from 20 isolated Among isolates, maximum resistance was observed carried this gene and showed bands after against ampicillin (100%) and amoxicillin/clavulanic acid electrophoresis examination. Certain strains that secrete (100%), followed by kanamycin (33.33%) and then large amounts of SEA, regardless of the environment, are tetracycline (6.66%). A high degree of susceptibility was likely to contribute to increased food safety risks. observed against chloramphenicol, streptomycin,

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Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat Figure 9. see= Staphylococcus aureus see PCR amplification of enterotoxin genes ( 270bp) of : L-DNA ladder 100bp, lanes DISCUSSION (1,3,5,6,8)) positive isolates. While lanes (2,4,7,9) negative isolates for egteanle. . S. aureus S. apurerveuiosus studies by Bantawa (2018), they showeetdal. Meat microbial spoilage is a complex happening that in 68% samples [48]. While, the prevalence of many different bacterial groups may contribute to, in meat wSa.sahuirgehuesr than the study of Rong depending on storage temperature and packing (20S1. 7a)u,rtehues results of them showed 37.2% of samples conditions. Damage can result from microbial were positive for [49]. The increased prevalence development and consumption of meat feeders by of refers to insufficient cleaning, unsatisfactorSy. bacteria, resulting in the release of unwanted metabolites ahuanredulisng, and post-pollution contamination from [40]. Food-borne pathogenic microbes are considered the contaminated air around stores. A high percentage of leading cause of disease and death in developing in uncooked meat and processors has health risks countries, causing heavy losses in health care. In additionE,. such as -mediated virulence and consumer invasion cmoolire andSmalomreonaetltleantion has recently been paid to food [50]. safety, especially after the outbreak of diseases due to S.tapahuyrleoucsocci are colonized on the skin and nose and and , among other causes, as well as easily transferred to the food causing food contamination. attention to eating raw and unpasteurized foods [41]. The could be able to produce toxins called World Health Organization has estimated that 1 in 10 Staphylococcal enterotoxins, these toxins are people are exposed to the disease and about 4 million and characterized by resistant to several conditions such as 20 thousand Spaelompolenedlliae eEascheyriecahriaasStaaprheysluolctooccfuesating proteolytic enzymes and heat, therefore they resistant the Lcoisntetarmiainated food. The bacteria that cause disease in and to the gastrointestinal tract of the human food, such as , , , and [51]. The existence of these pathogens is a major concern are considered a real danger to human health because some strains are able to produce stable heat [42]. During the logarithmic phase of bacterial growth, enterSottaopxhinyslocthocactucsaauusreeufsood poisoning in people, and bacteria are existing on the surface of the meat. When the therefore must be taken into account in risk valuation protein-degenerating bacteria (proteolytic bacteria) [52]. is one of the leading causes of approach the maximum cell density, the protease enzyme foodborneStdaipsehaysloecso. cTcuhse animal products such as milk secreted by the bacteria appears to break the connective and meat are often contaminated with enterotoxigenic tissue between the muscle fibers, allowing the bacteria to strains of . The contamination of food may penetrate the meat. While, Non-proteolytic bacteria do occur directly from infected food-producing animals or not penetrate meat, even when grown with proteolytic may consequence from poor hygiene throughout meat proteins [43]. Salmonella processes, or the storage of retail and food items, because In this study, the most prevalent isolates were isolated humans may carry microorganisms [53]. Staphylococcal from meat seatmapl.les are spp. at a perScaelnmtaognelolaf enteSrtaoptohxyilnosco(cScEuss) aruereausgroup of exotoxins, identified in 58%. This result was corresponding to the result of 1959 as extracellular proteins produced by some strains Bodhidatta (S2t0ap1h3y),lotchoecycuissolatedS8a4lm%onoeflla of . These toxins are known as from food samples [44]. pyrogenic toxins characterized by thermo-stable proteins The presence of and in meat that are resistant to many protein-degrading enzymes samples has an indication that the samples were such as pepsin, trypsin, chymotrypsinS,. aunrdeusrenin. contaminated from air, water, and hands while being virulence factors of staphylococci responsible for food slaughtered and cut in addition tᵒo that the toxins poisoning in humans [54]. Although are produced by these bacteria are thermostable and are not eliminated, toxins may remain and cause food poisoning eatffeaclted by temperature up to 100 C and for several [55]. Foodborne staphylococcal diseases acquired from hours [45]. this study agreed with tShaelmstoundeyllaof Bodhidatta eating food contaminated with inStaespthinylaolcotocxcuinss are the Arcob.,acthteery showed the moCsatmcpoymlombaocnterspecies isolated second most common kind of food-borne disease. The Sfraolmocnheillldaren with diarrhea were spp. (84%), high level of food poisoning with is due to (74%), and (51%) [44]. the inadequate pasteurization / disinfection of the source are one of foodborne pathogeSnailcmboancetlelaria that of the contaminated product [56]. causing severe diseases in human and animals, therefore, Foodborne diseases are the causes of illnesses and deaths the meat regarSdaeldmoanseallamain source of . Ye Y. Sinaldmeovneelollpaing countries and lead to the loss of billions of Saanldmhoniselclaoworkers showed 13% of meat samples were dollars in medical care, the most important oSfalwmhoincehllais positive for [46]. The transmitted of S,awlmhoicnhellcaauses foodborne diseases and food among humans andSaanlmimoanleslloaccurred during poisoning [57]. Meat is a major source of , consumption of contaminated food, therefore the detecting infection in Smalematonsealmlaples is very isolation and identification of formSalsmamonpellelas important to control and prevent foodborne diseases [58]. outbreak food-borne detection and prevent the entering Diarrheal diseases caused by are more the contaminated food into the food supply. spreading between children in developing countries can cause several different syndromes including comes after malaria then respiratory infections [59]. gastroenteritis, bacteremia, and typhoid fever, the most Several studies revealed the contamination of meat by common of which is gastroenteritis, whichetisals. ymptoms microorgaEn.ismcosliwhich canEbnetesruopcpooccrutesd ftaheiscasltiusdy, from like abdominal pain, diarrhea nausea, vomiting, and Swthaipchhy,lotchoeccsutsudayureoufs Sharma Saanlmd oCnhealltatosppapd. hyay they Shaelamdoacnheella[47]. Other study by BAodrchoibdatcttaer butz,lerreivealed isolated (98%), (9E0. %co)li, the mCaomstpycloombamcotenrly isolated pathogens from meat were (20%), (2%) and (84%Sta),pihnyalodcdoicticouns taoureus (74%) other organisms. They revealed high prevalence of and (51%) [44]. in meat products [60]. These results Saarlemaolnmeollsat identical In this study, was found in 38% to the results of our study but differ in the high from meat samples. This prevalence is lower than prevalence of bacteria in this study is . 472 Systematic Reviews in Pharmacy

Vol 11, Issue 11, Nov-Dec 2020 Characterization of Foodborne Pathogens and Enterotoxigenic Staphylococcus Aureus Isolates with Detection of Antibiotic Resistance from Beef Meat E. coli S. aureus

is the main bacterium that causes diseases that are of the isolates were confirmed to be . From them transmitted through food. Therefore, foods are preserved C42O.N4%CLwUSerIOe Ncarrying enterotoxin genes. in many ways, including chemical factors, for eEx.acmolpile the use of antibiotics, but they also generate bacteria that are resistant to antibiotics [61]. The isolated of Ef.rcoomli The results indicate the importance of continuous meat sample is a good indicator to intestinal monitoring of foodborne pathogens in butcher meat to contaminated during slaughtering process because reduce the risk of contamination. The availability of data regarded as normal flora of intestine [62]. on pathogenic microorganisms and their transmission in The differences in bacterial count among meat samples food from different countries would offer common due to the differences in raw materials and saneittaaryl. ground for reaching an internaEti.ocnoalli agreSeamlmenotneollna measures [63]. The result count of StapShtaylpohcyolcocciocacmuos ng regulations of food safety. The high load of Quarters was agreement with the study of Goja microorganisms and presence of and (2013), they reported4highest count of in indicates that the meat may be contaminated with SOtmapdhuyrlmocaonccu(s7.0a7u×r1e30us CFU/g) and lowest count in instinctual coSnatlemnotnaelnlad that consumers are at risk of Khartoum (4.46×10 CFU/g) [64]. The presence of developing a transmitted disease when ingested raw. The Shigella on meat samples may be detection of in meat, even in low-incidence contaminated from skin, nose, and nose of butchers. cases, has important implications. A Sstarlimctonpeulblalic health Only thSrheiegella was isolated from beef meat sample, and food safety system is urgently needed to reduce the similar to another result from Ethiopia which showed human health risks associated with disease. only 3 from 150 meat samples [65]. we conclude that our results showed the presence oSf. There are several reasons explain the increased of amuurletuidsrug-resistant bacteria in fresh meat and possibility bacteria in food production environments. Often times, of presence of enterotoxin genes in some isolates of circumstances in the food production environment are , suggesting that fresh beef meat products may act similar to those required for the product, such as as reservoirs of drug-resistant bacteria and faciSli.taatuerethues temperature, nutrients, and stress factors; Therefore, spread of resistance genes. Also, the existence of treatment surfaces may act as reservoirs for damaged enterotoxigenic and antimicrobial strains of bacteria. Additional, numerous studies have indicated bacteria has become noticeable in foods. This requires that the fate of pathogens introduced into the treatment better control over the sources of food contamination and environment may be affected by non-pathogenic bacteria AthCeKpNroOlWifeLrEatDioGnMoEfNaTntSibiotic-resistant organisms. [22]. Salmonella Resistant bacteria is a known public health problem, the results revealed that all isolates of were The authors would like to extend their sincere thanks to multidrug-resistant which mean resistance to four the University of Kufa and to the College of Sciences, antibiotics at least (streptomycin, amoxicillin, especially the Department of Biology, for their continued chloramphenicol, and gentamicin) [66]. When CsuOpNpFoLrItCinTpOrFodINucTiEngREthSiTs article. consumption of contaminated food with multi drug resistant food-borne pathogens will causes more serious diseases comparison with susceptible bacteria [62]. AThUeTaHuOthRoSrsCOdeNcTlaRrIeBtUhaTtItOhNere is no conflict of interest. Excessive use of antimicrobials and lack of hygiene during meat processing leading to undSeaslimraobnlellraisks to human health, for exampleStahlme opnreelslaence of leftovers of SA drafted the manuscript, compiled information from antimicrobial drugs, non-typhoidal , anSd. FthUeNlDitIeNraGture, and designed the figures and tables. WA anurtiemusicrobial resistance [67]. Multidrug reviewed and edited the manuscript. resistant bacteria, including methicillin-resistant , extended spectrum beta-lactamase DNoAnTeA. AVAILABILITY Enterobacteriaceae, and vancomycin-resistant Enterococci have a challenge to the human health care. These bacteria were detected in environment outside the All datasets generated or analyzed during this study are hospital, also found in meat and meat products [68]. EinTcHluIdCeSdSiTnAthTeEmMaEnNuTscript. Animals are usually administered with antibiotics in their feed, but thSetarpehiyslpocroecsecunst aatuterneutison in raising animals that are only administered with antibiotics throughout activSe. RNEonFeE.RENCES ainufreecutison. is a common pathogen for both humans and cattle raised for human ingesting. has attained high levels of antibiotic resistance, 1. Ahmed, S.A. and S. 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