ISOLATION AND CHARACTERIZATION OF ANTIBIOTIC SUSCEPTIBILITY PROFILE OF SALMONELLA SPECIES ISOLATED FROM ABATTOIR ENVIRONMENT

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1 Ife Jurnal f Science vl. 19, n. 2 (2017) ISOLATION AND CHAACTEIZATION OF ANTIBIOTIC SUSCEPTIBILITY POFILE OF SALMONELLA SPECIES ISOLATED FOM ABATTOI ENVIONMENT Igbinsa, E. O. * and Beshiru, A. Applied Micrbial Prcesses & Envirnmental Health esearch Grup, Department f Micrbilgy, Faculty f Life Sciences, University f Benin, P.M.B Benin City , Nigeria *Crrespnding authr: eigbinsa@gmail.cm st st (eceived: 31 August, 2017; Accepted: 21 Octber, 2017) ABSTACT In this study we investigated the antibigramic prfile f Salmnella species islated frm abattir envirnment. A ttal f 72 samples were cllected frm three different statins [statin A (the drainage), statin B (stagnant water in pthles and flrs arund the abattir) and statin C (water used fr washing the meat slaughtered at the abattir)] between January and June All samples were prcessed and analysed using standard culturebased and plymerase chain reactin (PC) methds. The mean ttal hetertrphic bacteria ppulatin densities were: statin A ± 0.02 CFU/ml; statin B ± 0.10 CFU/ml and statin C ± CFU/ml. The mean ttal salmnellae ppulatin density were ± 0.14 CFU/ml, ± CFU/ml, and ± 0.06 CFU/ml fr statin A, statin B and statin C respectively. A ttal f 50 cnfirmed salmnellae, using PC identificatin system, were subjected t antimicrbial prfiling using the disc diffusin methd. The resistance prfile f the islates revealed that 24 (48%) f the islates were resistant t piperacillin, 50 (100%) t gentamycin, 50 (100%) were resistant t tetracycline, while 22 (44%) f the islates were resistant t aztrenam. Multidrug resistance (MD) prfile f the islates revealed that 22 (44%) f the islates were resistant t piperacillin, gentamycin, tetracycline, and aztrenam belnging t fur antimicrbial class. Our findings revealed that abattir envirnments culd be a ptential reservir f multi-antibitic resistant Salmnella species. Keywrds: Abattir, Salmnellae, Antibigramic Prfile, Multi-Drug esistance, Effluent 389 INTODUCTION Salmnellsis is regarded as ne f the mst prevalent fdbrne zntic infectin in bth develped and develping cuntries, even thugh the prevalence seems t vary between cuntries (Mlla et al., 2003). It is usually challenging t explain the state f salmnellsis in develping cuntries resulting frm the very limited scpe f studies, with lack f c-rdinated epidemilgical surveillance systems. In additin, under-reprting f cases cupled with the presence f ther diseases well-thught-ut t be f high significance may have surpassed the prblem f salmnellsis in sme develping cuntries including Nigeria. The upsurge f glbal ppulace with mass prductin f animal and human fd as well as the rapid internatinal trade in aquaculture, agriculture and fd prducts culd further degenerate the prblem (Dahshan et al., 2010). Fd animals harbur a diverse range f salmnellae servar and thus, culd act as ptential reservir f cntaminatin, which is f significant epidemilgical imprtance in nntyphid human salmnellsis (Dallal et al., 2010). Cntaminatin f meat by Salmnella species may ccur at slaughterhuses frm animal faeces, symptmless animals, slaughter equipment, flrs as well as abattir persnnel. Interestingly, this pathgen usually gains access t meat at any stage during butchering and prcessing. Crsscntaminatin f carcasses and meat prducts culd cntinue during subsequent handling, prcessing, preparatin and distributin (Igbinsa, 2015a). The safety and hygienic quality f meat can be determined by the presence r absence f disease-causing micrrganisms. It has been reprted that fdbrne illnesses assciated with the cnsumptin f meat have cntinued t increase fr the past 30 years (Akachere et al., 2009). Salmnellae have been reprted frm abattir settings in different parts f Nigeria such as Osun State, (Omllu-As et al., 2017); Prt Harcurt, (Ire et al., 2017); Benue State, (Nseabasi-Maina et al., 2017); Skt, (Bagud et al., 2014; Faleke et al., 2017). Als frm ther Africa regins in

2 390 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella Nairbi, Kenya (Nyabundi et al., 2017); Kampala, Uganda (Tinega et al., 2016); Mansura, Egypt (Sallam et al., 2014) and Mdj and Bishftu, Ethipia (Kuma et al., 2017). Salmnella are amng rganisms currently under public health surveillance fr antimicrbial resistance (NAMS, 2013). An increasing number f primary surces f salmnellsis are cnsidered t have been linked t fd-prducing animals as well as cntaminated water surces (Kagambèga et al., 2013). Therefre, this study was cnducted t islate, identify and determine the antibigramic prfile f salmnellae frm abattir envirnment. MATEIALS AND METHOD Study area The abattir is situated at Ikpba slpe, lcated at latitude lngitude Benin City, Ed State, Nigeria. Ikpba slpe is a cmmunity clse t the Ikpba iver, a furth rder stream situated within the rainfrest belt f Ed State, Suthern Nigeria. The iver rises frm the Ishan Plateau in the Nrthern part and flwing in Suth Westerly directin in a steeply incised valley and thrugh sandy areas befre passing thrugh Benin City and jining the Ossim iver. Sample cllectin Seventy tw (72) wastewater samples were cllected within a six mnths perid (between January and June 2017), frm different sampling pints/lcatins at the abattir [statin A (the drainage), statin B (stagnant water in pthles and flrs arund the abattir) and statin C (water used fr washing the meat slaughtered at the abattir)]. All samples were cllected using sterile glass cntainer, labelled apprpriately and transprted t the Applied Micrbial Prcesses & Envirnmental Health esearch Grup Labratry, Department f Micrbilgy, Faculty f Life Sciences, University f Benin in cld ice pack and analysed within 24 h after cllectin. Enrichment and islatin prcedure Serial dilutin were carried ut where 0.1 ml f 6 8 each prepared dilutins (10-10 ) fr all wastewater samples was spread inculated int already prepared Nutrient Agar [NA] (Lab M, Lancashire, United Kingdm) plates in triplicates fr hetertrphic bacteria enumeratin; while 0.1 ml 5 f each prepared dilutins (10 ) fr all wastewater samples was inculated int sterile xylse lysine dexychlate (XLD) (Lab M, Lancashire, United Kingdm) Petri dishes in triplicates fr salmnellae enumeratin. Aliqut f 100 µl each were spread n the respective agar plate and allwed t absrb befre incubating at a temperature f 37 C fr h. After incubatin, discrete clnies were enumerated fr ttal hetertrphic bacteria and salmnellae cunt. The clur f salmnellae n the XLD agar plate was bserved as red with black centres. In additin, an aliqut f 1.0 ml frm each f the wastewater samples was inculated int test tubes cntaining 9.0 ml f Selenite F-Brth (Lab M, Lancashire, United Kingdm) and incubated fr h at 37 C. Thereafter, streak plate technique was emplyed by streaking directly frm the vernight culture frm Selenite F-Brth int a fresh XLD agar plate and incubated at 37 C fr h. ed clnies r red clnies with black centres was sub-cultured nt fresh XLD agar plates fr pure culture. Thereafter, distinct clnies were purified repeatedly n nutrient agar plates. Pure islates were stred n agar slants at 4 C fr further analysis. Phentypic identificatin f the Salmnella The purified islates frm culture-based technique using XLD agar medium (clnies that appear red, and usually with a black centre) were streaked n nutrient agar plates t btain pure cultures which were subjected t Gram-staining, xidase, urease reactins, indle and mtility tests. Organisms that appear as xidase negative, urease negative, indle negative and Gram-negative rds were selected presumptively as Salmnellae (Cheesbrugh, 2006). DNA extractin prcedure The genmic DNA f the presumptive salmnellae islates was extracted using biling methd. Distinct clnies f purified salmnellae islates were inculated int 200 µl f Tryptne Sy Brth (Merck, Darmstadt, Germany) and cultivated vernight at 37 C. The cells were lysed with heat blck (MK200-2, Shanghai, China) at 100 C fr 15 min. The biled bacterial cells were centrifuged at 14,500 rpm fr 10 min using micrcentrifuge t separate the cell debris frm the supernatant which was then stred at 20 C and

3 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella used as DNA template. Plymerase chain reactin identificatin using salmnellae genus-specific primer All PC amplificatin assay were cnducted in a reactin mixture (25.0 μl) with master mix (12.5 μl), frward (0.50 μl) and reverse (0.50 μl) primers, nuclease-free water (2.0 μl) and template DNA (5.0 μl). DNA templates f Salmnella enterica servar Typhimurium ATCC was used as a psitive cntrl while nuclease-free water as a negative cntrl in each PC assay. All PC reactins were carried ut using a Peltier-based thermal cycler (BiSeparatin System, Shanxi, China). The PC primer pair [F-5'-TGT TGT GGT TAA TAA CCG CA-3' and - 5'-CAC AAA TCC ATC TCT GGA-3'] amplicn size f 574 bp, fr the salmnellae genus-specific (16S rdna gene), and PC reactins were as described by Lin and Tsen (1996). PC prducts were electrphresed using 1.5% agarse gel (Hispanagar, Burgs, Spain) stained with 0.5 mg/l ethidium brmide (Merck, Mdderfntein, Suth Africa) at 100 V fr 1 h, in 1 TAE buffer and viewed under a UV trans-illuminatr (EBOX VX5, Vilber Lurmat, France). Antibitic susceptibility testing The Salmnella islates that were cnfirmed using the PC-based methd were subjected t antibigram characterizatin. Cmmercially available antibitics disc, btained frm Mast diagnstics, Merseyside, United Kingdm, were used t determine the resistance patterns f the islates against six different antibitics (1 dse/disc) gruped int six different classes f antibitics. Antibitic discs with the fllwing 391 drug cntents: piperacillin (100 µg), gentamycin (10 µg), aztrenam (30 µg), imipenem (10 µg), ciprflxacin (5µg) and tetracycline (30 µg) using the Kirby-Bauer disc-diffusin test, which cnfrms t the recmmended standard f the Clinical and Labratry Standards Institute (CLSI). These antibitics were chsen based n the treatment practices fr salmnellae in this area and frm the literature (Besser et al., 2000). The Salmnella were grwn fr 18 t 24 h n Mueller- Hintn brth (Merck, Darmstadt, Germany), harvested and then suspended in 0.85% nrmal saline slutin adjusted t a 0.5 McFarland 6 turbidity standard, crrespnding t 10 CFU/ml. The inculum was streaked nt plates f Mueller-Hintn agar using a sterile cttn swab and aseptically impregnated with the apprpriate antibitics. The results were recrded after 24 h f incubatin at 37 C. The diameter f the zne f inhibitin arund each disc was measured and interpreted as resistance (), intermediate (I) r sensitive (S) in accrdance with the recmmended standard established by the Clinical Labratry Standard Institute (2014). ESULTS Ppulatin density f the bacterial islates The mean hetertrphic bacterial cunt are ± 0.02 CFU/ml frm statin A; ± 0.10 CFU/ml frm statin B; and ± 0.05 CFU/ml frm statin C. The mean 5 salmnellae cunt are ± 0.14 CFU/ml 5 frm statin A; ± 0.15 CFU/ml frm 4 statin B; and ± 0.06 CFU/ml frm statin C (Table 1). Table 1: Mean f ttal hetertrphic and salmnellae cunt recvered frm abattir envirnment Ppulatin density Statins Min Max (CFU/ml) (CFU/ml) Mean ± SD (CFU/ml) Ttal hetertrphic bacteria Statin A ± 0.02 b Statin B ± 0.10 b Statin C ± 0.05 a p-value Salmnellae Statin A ± 0.14 b Statin B ± 0.15 b Statin C ± 0.06 a p-value Values are expressed in triplicates f mean ± standard deviatin. The mean difference are represented with superscript lwercase alphabets acrss clumn. Means with significant difference carry different alphabets (p<0.05).

4 392 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella A ttal f 78 islates f salmnellae were islated using the culture-based methds. Further classical characterizatin f the islates using mrphlgical and bichemical reactins, revealed that 72/78 (92.31%) islates were presumptively identified as salmnellae and subjected t plymerase chain reactin using salmnellae genus-specific primer, a ttal f 50/72 (69.44%) salmnellae were cnfirmed as Salmnella species. esistance prfile f the bacterial islates The resistance prfile f 50 Salmnella species identified frm abattir settings were further phentypically characterized using six (6) antibitics which are representatives f six (6) different grups f antimicrbial agents. The resistance prfile f the islates revealed that 24/50 (48%) f the islates were resistant t piperacillin; 50/50 (100%) were resistant t gentamycin; 50/50 (100%) were resistant t tetracycline; 4/50 (8%) were resistant t imipenem; 4/50 (8%) were resistant t ciprflxacin; 22/50 (44%) f the islates were resistant t aztrenam (Table 2). The distributin f antibitics susceptibility prfile f the islates frm abattir shwed that 16/18 (88.89%) and 8/16(50%) were resistant t the activity f piperacillin in statin A, and statin B respectively (Table 3). Als gentamycin and tetracycline were 100% resistant acrss the sampling statins during the study regimen (Table 3). Table 2: Antimicrbial agents, disc cntent and zne diameter interpretative standards and resistance prfile f Salmnella spp. frm abattir envirnment Antibitics Grup Antimicrbial Agent Disc cntent Zne diameter (mm) esistant Salmnellae n=50 (%) esistant Intermediate Sensitive Penicillins Piperacillin 100 µg ³ 21 24/50 (48) Aminglycsides Gentamycin 10 µg ³ 15 50/50 (100) Mnbactams Aztrenam 30 µg ³ 21 22/50 (44) Carbapenems Imipenem 10 µg ³ 23 4/50 (8) Flurquinlnes Ciprflxacin 5 µg ³ 31 4/50 (8) Tetracyclines Tetracycline 30 µg ³ 15 50/50 (100) Table 3: Distributin f the resistant prfile f Salmnella spp. frm abattir envirnment Antimicrbial agents Statin A n=18 (%) Statin B n=16 (%) Statin C n=16 (%) p-value Piperacillin (100 µg) 16 (88.89) 8 (50) 0 (0) Gentamycin (10 µg) 18 (100) 16 (100) 16 (100) Aztrenam (30 µg) 10 (55.55) 6 (37.5) 6 (37.5) Imipenem (10 µg) 2 (22.22) 0 (0) 0 (0) Ciprflxacin (5 µg) 2 (11.11) 2 (12.5) 0 (0) Tetracycline (30 µg) 18 (100) 16 (100) 16 (100) Multidrug resistance (MD) prfile f the islates revealed that 24/50 (48%) f the islates were resistant t PTZ, GEN and TET ; while 22/50 (44%) f the islate was resistant t PTZ, GEN, TET, and ATM. Extensive drug-resistant prfile f the Salmnella spp. revealed that 50/50 islates were resistant t GEN and TET (Table 4). Multiple antibitic resistant (MA) index revealed an index that ranged frm 0.3 t 1; shwing resistance t a minimum f 2 antibitics and a maximum f 6 antibitics.

5 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella Table 4: Multidrug-resistant and extensive drug-resistant prfile f Salmnella spp. islated frm abattir envirnment 393 Number f antimicrbial grup Number f antibitics esistance phentype Number f islates n=50 (%) 6 6 IMI, CIP, ATM, TET, GEN, PTZ PTZ, GEN, ATM, TET PTZ, GEN, TET GEN, TET Multiple antibitic resistant (MA) index Legend: PTZ: Piperacillin, ATM: Aztrenam, TET: Tetracycline, GEN: Gentamycin, IMI: Imipenem, and CIP: Ciprflxacin. DISCUSSION Salmnellae infectin can lead t invasive and fcal infectins that can be severe. The ability f Salmnella t cause invasive infectin varies with the servar. Gd persnal and envirnmental hygienic practices are instrumental in fd safety. They are cmpulsry by law under the auspices f internatinal and natinal hygiene prtcls and are ften described as prerequisites t fd safety guidelines based n Hazard Analysis and Critical Cntrl Pint (HACCP). Pr hygiene in abattir envirnment almst always results in disseminatin and prliferatin f pathgenic clnes as well as fd spilage micrbes n the envirnments f slaughterhuses. In the present study we examined the presence f antibiticresistant Salmnella species frm abattir envirnments using classical and PC tls. A significant difference in the hetertrphic bacteria as well as the salmnellae cell densities frm the different sampling pints was bserved (p < 0.05). The high ppulatin f hetertrphic bacteria and salmnellae cunts bserved in this study culd directly be attributed t the fact that the abattir envirnments were cntaminated due t pr persnal and envirnmental hygiene f the butchers wh slaughter meat at the abattir; their waste discharged arund the abattir thereby making the meat unsafe fr cnsumptin; and favuring the prliferatin f antibitic-resistant Salmnella spp., as bserved in this study. This culd als be attributed t the pr water quality in the abattir. The high rate f islatin f Salmnella frm statin A (drains) and statin B (pthles and flrs f the slaughterhuses) may be as a result f accumulatin f excreta frm cattle, prir t slaughter. There may als be sufficient nutrients in the mist envirnments t enable the prliferatin f Salmnella viability fr a lng duratin f time. It was bserved during this study that waste matter frm cattle was nt prperly remved regularly frm the abattir envirnments, as there was n effective cleaning prcedures put in place. The salmnellae in the envirnments culd cntaminate the cattle skin and hves, neighburing sils and water bdies resulting frm pr envirnmental hygiene as a cnsequence f accumulated faeces (dung). Mtsela et al. (2002) reprted that salmnellae were present in all sampled abattir envirnments in Btswana. Gragg et al. (2013) reprted the prevalence f Salmnella enterica subspecies enterica sertypes in cattle faeces amng ther surces in Mexic. Als, the ccurrence f Salmnella at variable frequency frm different samples btained frm gats (including faecal samples) in Ethipia has been dcumented (Wldemariam et al., 2005). There is every tendency that faeces frm the carcass, mixing with the meat, culd cntribute t the cntaminatin f the meat. This is als similar t the findings f Bell et al. (2011) in sme ther abattir in Nrth- Western Nigeria. In Nigeria, many abattirs dispse their effluents directly int streams and rivers withut any frm f treatment and the slaughtered animals are washed by the same water (Adelagan, 2002). This is the case in mst abattirs in Nigeria and we bserved similarity during ur sampling regime. Therefre, the ccurrence f high ttal hetertrphic bacteria and salmnellae cunt in the effluent frm this abattir implies a lt fr public health. Cntaminatin f abattir surfaces may emanate frm the butchers, which culd be as a result f using unglved hands during dressing and prcessing f the carcass in the slaughtering hall. In additin, the ftwears used by the butchers culd functin as a vehicle fr faecal

6 394 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella cntaminatin, since prcessing and dressing f the carcasses ccurs n the pen flr in the slaughterhuses. Mrever, in develping cuntries like Nigeria, water bdies such as river are used fr drinking, bathing, washing, watering f crps and ther dmestic purpses which als indicate a high impact n the public health f the users (Nafarnda et al., 2012). Similar study cnducted by Mr-Mur and Yuste (2010) indicated that bacteria islated frm wastes and abattir prducts include pseudmnads, salmnellae, klebsiellae and Prteus. Salmnella islates btained in the study were multi-drug resistant. The islates frm the abattir envirnments (statin A, B and C) were fund t demnstrate variable resistance patterns. Multidrug resistance (MD) has been described as acquired resistance t at least ne antimicrbial agent in three r mre antimicrbial classes (i.e. bacterial islates remain resistant t 1 antimicrbial in 3 antimicrbial categries) while extensively drug-resistant (XD) has been described as nn-susceptibility t 1 antimicrbial agent in 2 antimicrbial classes (Magiraks et al., 2012). This frmed the basis fr MD and XD prfile f islates in this study. A significant regressin (r = ) f the number f islates n the resistance phentype was bserved (p < 0.05). This culd be as a cnsequence f the disseminatin f antimicrbial resistant genes within bacterial ppulatin via hrizntal gene transfer. Similarly, intrductin f such multidrug-resistant pathgens in the envirnment and subsequent accumulatin uphlds the spread f resistant pathtypes thrugh transfer f integrns and plasmids within micrbita resulting in the frmatin f a pl f resistance reservir. It has been reprted that antibitics are widely used in fd animal prductin fr therapy and preventin f bacterial infectin and fr grwth prmtin (Igbinsa et al., 2016). Studies have als shwn that 50-90% f drugs administered t farm animals are excreted int the envirnment either un-metablized r as metablic intermediates which even thugh inactive, may underg transfrmatin t the active frm in the envirnment (Dahshan et al., 2010; Cabral et al., 2017). In the curse f this study, it was bserved that discharge f faecal material frm the prcessing carcasses was being released as effluent in the envirnment. This can cnsist f a lt f un-metablized antibitic residue (Kummerer, 2003). Findings frm Prendergast et al. (2007) clearly demnstrate the ptential fr crsscntaminatin frm equipment and meat cntact surfaces in the cutting rm envirnment. Multidrug resistant Salmnella species have been elucidated by previus researchers (Gmes-Neves et al., 2014; Sallam et al., 2014; Nseabasi-Maina et al., 2017). On sme ccasin, the same sertype frm a single animal was islated frm mre than ne sample (i.e. faeces and rumen; faeces and carcass; rumen and carcass; faeces, rumen and carcass) (McEvy et al., 2003). esistant micrrganisms can persist in waste, sil, fd and water with a number f cnsequences (Igbinsa, 2015a; 2015b). The ccurrence f multi-drug resistant (MD) Salmnella spp., culd pse majr public health risks t cnsumers. Firstly, ingestin f prducts cntaminated with MD Salmnella species culd cause salmnellsis which requires antibitic therapy and therapy can then be cmprmised due t resistant strain (Omllu-As et al., 2017). Hwever, it shuld be nted that even if the prducts are cked, it will nt destry genes respnsible fr the resistance. Secndly, resistant nn-pathgenic bacteria can be transferred t human via cnsumptin f cntaminated fd prducts and resistance genes are subsequently transferred t ther bacteria in the gut thrugh mbile genetic element such a plasmid, integrns and gene cassette, insertin sequence and transpsn (Mbt et al., 2012; Bagudu et al., 2014). Lastly, antibitics which may remain as residues in animal prducts such as meat, liver and bld can als lead t the selective prliferatin f resistant clnes in the cnsumer f the prduct (Egan et al., 2017). The islating rate f salmnellae frm abattir envirnment is increasing with a multidrug resistance and an increasing tendency f their resistance rates. Therefre, it is necessary t imprve bacterial resistance mnitring and antibigram research t direct ratinal drug therapy.

7 Igbinsa and Beshiru: Islatin and Characterizatin f Antibitic Susceptibility Prfile f Salmnella CONCLUSION We cnclude frm ur study that effluents frm the abattir culd be a ptential surce f reservir and disseminatin f antibitic-resistant salmnellae between animal and human ppulatin as well as the envirnment. This calls fr surveillance netwrk system t track resistance patterns f salmnellae servars circulating in abattir and ther envirnmental surces. Implementing suitable Hazard Analysis and Critical Cntrl Pints (HACCP) prcedures t decrease the crss-cntaminatin frm these envirnmental surces as well as fd animal handlers and the final cnsumers culd decrease the ccurrence f multiple antibitic resistant salmnellae in the envirnment. Cnflict f Interest The authrs declare n cnflict f interest EFEENCES Adelagan, J.A Envirnmental plicy and slaughter huse waste in Nigeria. In: Prceedings f the 28th WEDC Cnference, India. Akachere, J.T.K., Tanih, N.F., Ndip, L.M. and N d i p,. N P h e n t y p i c characterizatin f Salmnella typhimurium islates frm fd-animals and abattir drains in Buea, Camern. J. Health Ppulatin Nutr. 27: Bagud, A.I., Tambuwal, F.M., Faleke, O.O., Egwu, O.O. and Alier, A.A Prevalence f Salmnella sertypes in Skt abattir effluents and vegetables cultivated arund the abattir. Micrbil. es. Int. 2(2): Bell, M., Lawan, M.K., Kwaga, J.K. and aji, M.A Assessment f carcass cntaminatin with E. cli O157 befre and after washing with water at abattirs in Nigeria. Int. J. Fd Micrbil. 150: Besser, T.E., Gldft, M., Pritchett, L.C., Khakhria,., Hancck, D.D., ice, D.H., Gay, J.M., Jhnsn, W. and Gay, C.C Multiresistant Salmnella Typhimurium DT104 infectins f humans and dmestic animals in Pacific Nrthwest f the United States. Epidemil Infect. 124: Cabral, C.C., Panzenhagen, P.H.N., Delgad, K.F., Silva, G..A., drigues, D.D.P., Franc, 395. M. a n d C n t e, C. A Cntaminatin f carcasses and utensils in small swine slaughterhuses by Salmnella, in the Nrth-Western regin f i de Janeir, Brazil. J. Fd Prtect. 80(7): Clinical and Labratry Standards Institute, Perfrmance standards fr antimicrbial susceptibility testing; twenty-furth internatinal supplements. Wayne: Clinical and Labratry Standards Institute. Cheesbrugh, M Cheesbrugh M, editr. District labratry practice in trpical cuntries, pt 2. 2d ed. Cambridge: C a m b r i d g e U n i v e r s i t y P r e s s ; Micrbilgical tests. Chapter 7; pp Dahshan, H., Shahada, F., Chuma, T., Mriki, H. and Okamt, K Genetic analysis f multidrug-resistant Salmnella enterica servars Stanley and Typhimurium frm cattle. Vet. Micrbil. 145: Dallal, M.M.S., Dyle, M.P., ezadehbashi, M., Dabiri, H., Sanaei, M., Mdarresi, S., Bakhtiari,., Sharifiy, K., Taremi, M., Zali, M.. and Sharifi-Yazdi, M.K Prevalence and antimicrbial resistance prfiles f Salmnella sertypes, Campylbacter and Yersinia spp. islated frm retail chicken and beef, Tehran, Iran. Fd Cntrl 21: Egan, D.A., Naughtn, V., Dley, J.S. and Naughtn, P.J Detectin f Salmnella enterica servar issen in slaughter Pigs in Nrthern Ireland. Adv. Micrbil. 7: Faleke, O.O., Jlayemi, K.O., Igh, Y.O., Jibril, A.H. and Ayedun, G.O Salmnella species n meat cntact surfaces and prcessing water in Skt main market and abattir, Nigeria. Macednian Vet. ev. 40(1): Gmes-Neves, E., Antunes, P., Manageir, V., Gartner, F., Canica, M., da Csta, J.M.C. and Peixe, L Clinically relevant multidrug resistant Salmnella enterica in swine and meat handlers at the abattir. Vet. Micrbil. 168: Gragg, S.E., Lneragan, G.H., Nightingale, K.K., Brichta-Harhay, D.M., uiz, H., Jacb,.,

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