Veterinry World, EISSN: 2231-0916 RESEARCH ARTICLE Open Access Sources of contmintion, prevlence, nd ntimicrobil resistnce of thermophilic Cmpylobcter isolted from turkeys Rdi Bouhmed 1, Leil Bouyd 1, Sr Messd 1, Sfi Zeni 1, Mlek Nïm 2 nd Th-Mossdk Hmdi 1 1. Lbortory of Food Hygiene nd Qulity Insurnce System, High Ntionl Veterinry School, Rue Issd Abbes, 16111 El Ali, Oued Smr, Algiers, Algeri; 2. Deprtment of Microbiology, Centrl Militry Hospitl, 16050 Koub, Algiers, Algeri. Corresponding uthor: Rdi Bouhmed, e-mil: bouhmed.r@gmil.com Co-uthors: LB: leil_bouyd@hotmil.com, SM: srmessd@hotmil.com, SZ: sfi_zeni@yhoo.fr, MN: nimlek@hotmil.com, TMH: mousshmdi@hotmil.com Received: 02-05-2018, Accepted: 05-07-2018, Published online: 07-08-2018 doi: 10.14202/vetworld.2018.1074-1081 How to cite this rticle: Bouhmed R, Bouyd L, Messd S, Zeni S, Nïm M, Hmdi TM (2018) Sources of contmintion, prevlence, nd ntimicrobil resistnce of thermophilic Cmpylobcter isolted from turkeys. Veterinry World, 11(8):1074-1081. Abstrct Aim: Sources of contmintion, prevlence, nd ntimicrobil susceptibility of thermophilic Cmpylobcter isolted from turkey smples were determined. Mterils nd Methods: A totl of 300 smples were collected from 3 frms (fecl droppings) nd 4 poultry slughterhouses (neck skins nd cec) locted in the middle re of Algeri (Algiers, Boumerdès, nd Bouir). After detection, n ntibiogrm ws relized only for slughterhouses smples. Results: Smples from cecum (90.0%, 90/100; 95% confidence intervl (CI)=84.1-95.9%), fecl dropping (68.0%, 68/100; 95% CI=58.9 77.1%), nd neck skin (55.0%, 55/100; 95% CI=45.2 64.8%) were positive for thermophilic Cmpylobcter (p<0.05). Contmintion rte of turkey crcsses ws higher in modern slughterhouse (96.7%) thn in trditionl slughterhouses (37.1%) (p<0.05). Isolted strins were resistnt to nlidixic cid (NA) (87.5%), tetrcycline (TE) (81.3%), ciprofloxcin (CIP) (75.0%), mpicillin (AM) (65.6%), nd erythromycin (25.0%) (p<0.05). 96.9% (124/128) of the isoltes were multiresistnt nd 18 drug resistnce ptterns were registered. The predominnt one (43.0%) ws AM, NA, CIP, nd TE. Conclusions: Potentil sources of contmintion of this fstidious bcterium were noticed in frms nd slughterhouses. Modern slughterhouse llowed contmintion of turkey crcsses more thn trditionl slughterhouse. However, the sclding step could not represent source of contmintion. The most tested strins exhibited resistnce to erythromycin nd/or CIP. It is worrisome becuse these molecules re considered s first-choice ntibiotics for humn cmpylobcteriosis. Keywords: ntimicrobil resistnce, frm, slughterhouse, Thermophilic Cmpylobcter, turkey. Introduction Cmpylobcter is considered worldwide s the mjor cuse of gstroenteritis in humns [1]. In 2010, 109,700 ftl cses worldwide were reported. The disese rte in developing countries is 400 to 600 per 100.000 mong children <5 yers of ge. For developed countries, the disese rte is 300 per 100.000. In both developing nd developed countries, rtes in the generl popultion re estimted t 90 per 100.000 [2]. Humn cmpylobcteriosis occurs minly following consumption of contminted rw or undercooked food or contminted wter [1]. Furthermore, due to the gut coloniztion by thermophilic Cmpylobcter of nimls intended for humn consumption, met contmintion hs minly digestive origin, nd it occurs during slughtering [1,3]. Copyright: Bouhmed, et l. Open Access. This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License (http://cretivecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver (http:// cretivecommons.org/publicdomin/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted. However, mong the entire foodstuff, poultry met notbly broiler nd turkey re considered to be the min vehicle of thermophilic Cmpylobcter to humn [4]. A low infectious dose is enough to cuse ordinry Cmpylobcter enteritis which frequently progresses to hemorrhgic enteritis nd sometimes even to Guillin-Brre syndrome [1,3]. In generl, the ptient eventully hels without resorting to ntibiotic tretment, but in severe cses, ntibiotic tretment is needed [5]. Thermophilic Cmpylobcter both in nimls nd humns hve cquired over time resistnce to vrious ntibiotics, including erythromycin, nd ciprofloxcin (CIP), the mjor molecules for the tretment of Cmpylobcter infection [6]. Thus, the presence of Cmpylobcter strins resistnt to ntibiotics in foodstuffs of niml origin represents sig nificnt thret to public helth [5]. In Algeri, previous study reported tht thermophilic Cmpylobcter ws considered s cuse of humn gstroenteritis. Indeed, they were isolted from humn fecl smples with rte of 17.7% [7]. In developed countries, thermophilic Cmpylobcter isoltion rtes rnged from 55 to 77% in turkey smples [8,9]. Veterinry World, EISSN: 2231-0916 1074
To the best of our knowledge, there re no published dt on the presence of thermophilic Cmpylobcter in Algerin turkey smples, even if turkey met is frequently consumed by our popultion. Therefore, the current study ws crried out to determine sources of contmintion, the incidence of thermophilic Cmpylobcter on turkey in both poultry frms nd poultry slughterhouses, nd to study the ntibiotic susceptibility of Cmpylobcter strins isolted from poultry slughterhouses smples. Mterils nd Methods Ethicl pprovl In this study, we used turkey droppings, smples from cecum nd neck skin of turkey crcsses. Therefore, no ethicl pprovl ws needed. Smples collection Before ech smpling, questionnire ws filled out through the informtion provided by the owner nd in some cses by the veterinrin of the estblishment. 300 smples were collected septiclly nd rndomly from 7 estblishments locted in the region of Algiers: 3 frms nd 4 poultry slughterhouses. This llowed us to hve smples from vrious loctions nd regions. During the end of the rering period, totl of 100 smples of fecl droppings were collected 1-2 weeks before removl in three turkey frms (A, B, nd C). In ddition, 100 smples ech of neck skins nd cec were collected just fter turkey s eviscertion within 4 poultry slughterhouses (three trditionl [A, B, nd C] nd one modern [D]). With livestock cpcity rnging from 800 to 900 subjects, the visited frms were locted in rurl res including subjects delivered from the sme htchery nd rered in single bnd until slughter. The visited slughterhouses were locted in urbn or industril res, nd their cpcity rnged from 300 to 700 birds per week. All the smples were tken erly in the morning within 2 h. 1-2 weeks before slughter, fresh fecl smples were collected from litters using sterile sptuls. We pid ttention to collect them quickly nd septiclly just fter their emission on the ground without ny trce of litter or urine. At present, there is not n ccepted stndrd method for the detection nd isoltion of Cmpylobcter spp. t frm level [10]. We decided not to choose clocl swbs becuse we followed the OIE mnul which recommends to collect freshly voided feces (>10 6 UFC/g) for relible detection of Cmpylobcter by culture [11,12]. Furthermore, ccording to some uthors, isoltion from fecl smples give better results thn isoltion from clocl swbs [13]. During the slughtering process, just fter the eviscertion step, neck skins nd cec were septiclly collected. Fecl dropping nd cecum smples were put in seprte sterile universl plstic smple continers, while neck skins were plced in seprte sterile plstic bgs. After tht, ll the smples were plced inside cool box nd trnsported immeditely to the Deprtment of Microbiology of the Centrl Militry Hospitl of Algiers where they were processed within 2-4 h. Detection of thermophilic Cmpylobcter All the Cmpylobcter cultures (isoltion, identifiction, nd ntimicrobil susceptibility testing) were obtined with the microerophilic genertors GENbgmicroer (biomérieux) (5% O 2, 10% CO 2, nd 85% N 2 ). Identifiction of thermophilic Cmpylobcter From ech smple of feces or cecl content, 1g ws inoculted into 9 ml of sterile sline (0.9% NCl, w/v) nd homogenized. For neck skins, 10 g of ech neck skin ws dded in 90 ml of Preston broth (Oxoid) with 5% horse blood (IPA: Institut Psteur d Algérie) nd incubted t 42 C for 24 h. An gr Cmpylosel redy to use (biomérieux) for droppings smples, nd Butzler gr (Oxoid) with 5% horse blood (IPA) for neck skin smples nd cecl contents, were seeded nd incubted t 42 C for 48 h in the microerophilic tmosphere [14]. Confirmtion of the suspected colonies Once purified onto Columbi gr (Bio Rd) with 5% horse blood (IPA), identifiction of Cmpylobcter strins ws performed using the stndrd tests: Grm stin, motility, ctlse, nd oxidse rections, growth t 25 C nd erobic growth. Suspected colonies of Cmpylobcter were subjected to confirmtion by studying biochemicl tests on triple sugr iron gr (IPA) nd by testing their sensitivity to nlidixic cid (NA) (30 µg) nd cephlothin (KF) (30 µg) [11,15]. After tht, only one strin from ech Cmpylobcterpositive smple ws selected for susceptibility testing. Antimicrobil susceptibility testing Determintion of ntibiotic susceptibility of Cmpylobcter ws relized only for strins isolted from smples tht were collected from poultry slughterhouses, following the disk diffusion method s recommended by the ntibiogrm committee of the French Society of Microbiology [16]. In ddition to NA nd KF, the tested ntibiotics were: Ampicillin (AM) (10 µg), gentmicin (15 µg) (GM), erythromycin (15 UI) (E), CIP (5 µg), tetrcycline (30 UI) (TE), nd chlormphenicol (30 µg) (C). From pure culture of 18-24 h of incubtion, bcteril suspension of 0.5 McFrlnd opcity ws prepred nd diluted 1:10. After seeding by swbbing on Mueller-Hinton gr (Bio-Rd) contining 5% horse blood (IPA), nd pplying discs of ntibiotics (biomérieux), the pltes were in the microerophilic tmosphere during 24 h t 37 C. The dimeters of inhibition zones were mesured using metl cliper. For qulity control, the reference strins Escherichi coli ATCC 25922 nd Stphylococcus ureus ATCC 25923 were used. Sttisticl nlysis Chi-squre nd Fisher s exct tests were performed to compre the results of the tested smples nd the ntimicrobil susceptibility testing. The difference Veterinry World, EISSN: 2231-0916 1075
ws significnt when the p<0.05. Furthermore, 95% confidence intervl (95% CI) ws lso determined for contmintion nd resistnce rtes. Results Sources of contmintion In ll the estblishments host of risk fctors were observed. The results of frm nd slughterhouse surveys re reported in Tble-1. Most frms were mixed houses (66.7%) where fresh litter ws used (66.7%), ccess of wild or domestic nimls in the houses ws not controlled (100.0%), turkey feces were used s mnure to fertilize crops (100.0%), nd drinking wter ws dirty nd contined fethers (100.0%). In slughterhouses, severl sources of contmintion such s the bsence of mintining forwrd movement (0.0%), fixed sttion (0.0%), steriliztion of the slughtering equipment (0.0%), clening nd/ or disinfection protocol (0.0%), nd presence of dirty uniforms (75.0%) were observed. Detection of thermophilic Cmpylobcter Thermophilic Cmpylobcter strins were isolted with high prevlence in ll the smpled turkey flocks (71.0% nd 213/300) in both frms (68.0% nd 68/100) nd slughter fcilities (72.5% nd 145/200) (Tble-2). In totl, they were detected in 90.0% (95% CI = 84.1-95.9%) of cecl contents, 68.0% (95% CI = 58.9-77.1%) of fecl droppings, nd 55.0% (95% CI = 45.2-64.8%) of neck skins (Tble-2). The difference between these results ws sttisticlly significnt (p<0.05). Furthermore, for neck skin smples, the overll prevlence of thermophilic Cmpylobcter isolted from trditionl slughterhouses (37.1%, 26/70; 95% CI=25.8-48.5%) ws lower thn the one registered in modern slughterhouse (96.7%, 29/30; 95% CI=82.8-99.9%) (p<0.05). However, thermophilic Cmpylobcter ws isolted with high rtes from cecl contents in both trditionl nd modern slughterhouses s whole (88.6%, 62/70 vs. 93.3%, 28/30; p>0.05). Tble-1: Potentil sources of Cmpylobcter trnsmission in frms nd slughterhouses. Generl chrcteristics Frms Totl n (%) A B C Mixed flock (broiler+turkey) + + 2/3 (66.7) Other livestock Rbbits nd bees cttle 2/3 (66.7) Other nimls (dogs, cts, wild birds, nd rodents) + + + 3/3 (100.0) Insects Flies Flies 2/3 (66.7) Litter Spent (moist) Fresh (dry) Fresh (dry) 2/3 (66.7) b Wter qulity control 0/3 (0.0) Drinking wter Dirty Dirty Dirty 3/3 (100.0) Pest control Rts nd insects Rts 2/3 (66.7) Clening nd/or disinfection protocol 0/3 (0.0) Using turkey feces s mnure + + + 3/3 (100.0) Slughterhouses A B+C D Respect of trnsport conditions (from flock to slughterhouse) 0/4 (0.0) Respect of feed withdrwl+rest period + 2/4 (50.0) Mixed slughterhouse (broiler+turkey) + + 2/4 (50.0) Slughtering process Mnul Mnul Industril 3/4 (75.0) c Mintining forwrd movement 0/4 (0.0) Fixed sttion 0/4 (0.0) Respect of the sclding wter temperture + 1/4 (25.0) Steriliztion of the slughtering equipment 0/4 (0.0) Clening nd/or disinfection protocol 0/4 (0.0) Worker s uniform Dirty Plus wild bor, b Fresh litter (dry), c Mnul Dirty + Clen 1/4 (25.0) 3/4 (75.0) Tble-2: Prevlence of thermophilic Cmpylobcter in the visited frms nd poultry slughterhouses. Frms Flock Fecl droppings Number /exmined smples (%) Flock Cecl content Number /exmined smples (%) Slughterhouses Neck skin Number/exmined smples (%) A 29/35 (82.9) A 16/20 (80.0) 6/20 (30.0) B 17/32 (53.1) B 19/20 (95.0) 13/20 (65.0) C 22/33 (66.7) C 27/30 (90.0) 7/30 (23.3) Totl 68/100 (68.0) D 28/30 (93.3) 29/30 (96.7) Totl 90/100 (90.0) 55/100 (55.0) Significnt difference (p<0.05) between the results of cecl contents nd neck skins Veterinry World, EISSN: 2231-0916 1076
Antimicrobil susceptibility testing Antimicrobil susceptibility testing ws studied for 128 of 145 isolted strins from poultry slughterhouses becuse 17 thermophilic Cmpylobcter strins were impossible to re-strek (vible but non-cultivble form). 87.5% (n=112; 95% CI=81.8-93.2) strins were resistnt to NA, 81.3% (n=104; 95% CI=74.5-88.0) to TE, 75.0% (n=96; 95% CI=67.5-82.5) to CIP, 65.6% to AM (n=84; 95% CI=57.4-73.9), nd 25.0% (n=32; 95% CI=17.5 32.5) to E, nd no resistnce ws recorded for GM nd chlormphenicol (C) (0.0%) (p<0.05) (Tble 3). The study of the ntimicrobil susceptibility of TTC ccording to the type of smples reveled tht the difference between the rtes of ech tested ntibiotic for strins isolted from cecl contents nd neck skins ws not sttisticlly significnt (p>0.05) (Tble-3). Furthermore, rtes of resistnce to NA, E, CIP, TE, nd AM between ll the visited slughterhouses (A, B, C, nd D) were significntly different (Tble-4). Multidrug-resistnt Cmpylobcter isoltes were common. All the tested strins were resistnt to t lest one ntibiotic (100.0%), 124 of 128 tested strins (96.9%) were multidrug-resistnt. 17.2% (n=22) isoltes were resistnt to two ntibiotics, 25.0% (n=32) to three ntibiotics, 51.6% (n=66) to four ntibiotics, nd 3.1% (n=4) to five ntibiotics. Furthermore, 18 drug resistnce ptterns were identified, nd the most prevlent multiple resistnce profiles were observed for 55 isoltes (43.0%) nd included AM, NA, TE, nd CIP. 17 ntimicrobil resistnce ptterns for cecl contents isoltes nd 9 ntimicrobil resistnce ptterns of thermophilic Cmpylobcter strins isolted from neck skins were noticed. Resistnce to CIP nd/or E ws found in 85.9% of the isoltes tht were multiresistnt (n=110). Results of resistnce ptterns of thermophilic Cmpylobcter isoltes (n=124) tht were resistnt to two or more ntibiotics re reported in Tble-5. Discussion Except the influence of selective culture medi nd sources of contmintion noted on livestock, the significnt difference (p<0.05) between the rtes of Cmpylobcter contmintion of fecl smples nd cecl contents my lso depend on the smpling seson. Indeed, fecl droppings were smpled in winter while cecl contents were collected in summer. The pek of contmintion of poultry flocks by Cmpylobcter occurs in wrmer months [17,18]. The prevlence of thermophilic Cmpylobcter in fecl droppings ws in greement with previous study in Greece (77%) [19]. However, lower rtes (56%) hve been observed in the USA [9]. On turkey frms, these bcteri were present in t lest hlf of the fecl smples; which involves significnt horizontl trnsmission. Indeed, in ll the visited frms, we observed host of risk fctors (Tble-1) described for broiler flock coloniztion [18] such s exposure to potentil sources of the bcterium such s the presence of humns, other nimls (wild nd domestic nimls), insects nd rodents on frms, types nd qulity of litter, hving ccess to outside soil, nd using poultry feces s mnure. Contmintion from previous flocks my lso represent risk fctor [18]. Knowing tht to reduce the rte of Cmpylobcter in frms, high level of biosecurity control nd hygiene must be done [18] nd s there ws no clening nd/or disinfection protocol in ll the visited frms (Tble-1), Tble-3: Antimicrobil resistnce rtes of thermophilic Cmpylobcter strins ccording to the type of smples. Tested ntibiotics All the smples (n=128) Type of smples n (%) 95% CI b Cecl content n=81 (%) Neck skin n=47 (%) NA c 112 (87.5) 81.8 93.2 65 (80.3) 46 (97.9) TE c 104 (81.3) 74.5 88.0 63 (77.8) 41 (87.2) CIP c 96 (75.0) 67.5 82.5 59 (72.8) 37 (78.7) AM c 84 (65.6) 57.4 73.9 52 (64.2) 32 (68.1) E c 32 (25.0) 17.5 32.5 18 (22.2) 14 (29.8) GM 0 (0.0) 0 (0.0) 0 (0.0) C 0 (0.0) 0 (0.0) 0 (0.0) NA=Nlidixic Acid, TE=Tetrcycline, CIP=Ciprofloxcin, AM=Ampicillin, E=Erythromycin, GM=Gentmicin, C=Chlormphenicol, b 95% confidence intervl, c No significnt difference (p>0.05) between the results of cecl contents nd neck skins for ech tested ntibiotic Tble-4: Antimicrobil resistnce rtes of thermophilic Cmpylobcter strins ccording to the visited slughterhouses. Tested ntibiotics A n=17 (%) B n=29 (%) C n=27 (%) D n=55 (%) NA b 9 (52.9) 24 (82.8) 27 (100.0) 51 (92.7) TE b 15 (88.2) 14 (48.3) 21 (77.8) 54 (98.2) CIP b 12 (70.6) 7 (24.1) 22 (81.5) 55 (100.0) AM b 9 (52.9) 13 (44.8) 22 (81.5) 40 (72.7) E b 17 (100.0) 8 (27.6) 6 (22.2) 1 (1.8) GM 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) C 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) NA=Nlidixic cid, TE=Tetrcycline, CIP=Ciprofloxcin, AM=Ampicillin, E=Erythromycin, GM=Gentmicin, C=Chlormphenicol, b Significnt difference (p<0.05) between the results of the slughterhouses for ech tested ntibiotic Veterinry World, EISSN: 2231-0916 1077
Tble-5: Resistnce ptterns of thermophilic Cmpylobcter strins isolted from slughterhouses. No. ntimicrobils Pttern Isolted strins n=124 (%) Cecl contents n=78 (%) Neck skins n=46 (%) 2 AM NA 9 (7.3) 9 (11.5) 0 (0.0) 2 TE NA 5 (4.0) 4 (5.1) 1 (2.2) 2 CIP TE 3 (2.4) 3 (3.8) 0 (0.0) 2 CIP NA 2 (1.6) 1 (1.3) 1 (2.2) 2 E NA 2 (1.6) 1 (1.3) 1 (2.2) 2 AM CIP 1 (0.8) 0 (0.0) 1 (2.2) Totl 22 (17.7) 18 (23.1) 4 (8.7) 3 CIP TE NA 11 (8.9) 11 (14.1) 0 (0.0) 3 E TE NA 9 (7.3) 2 (2.6) 7 (15.2) 3 AM CIP NA 7 (5.6) 5 (6.4) 2 (4.3) 3 E CIP TE 2 (1.6) 2 (2.6) 0 (0.0) 3 E CIP NA 1 (0.8) 1 (1.3) 0 (0.0) 3 AM E TE 1 (0.8) 1 (1.3) 0 (0.0) 3 AM CIP TE 1 (0.8) 1 (1.3) 0 (0.0) Totl 32 (25.8) 23 (29.5) 9 (19.6) 4 AM CIP TE NA 55 (44.4) 28 (35.9) 27 (58.7) 4 AM E CIP TE 5 (4.0) 5 (6.4) 0 (0.0) 4 E CIP TE NA 5 (4.0) 1 (1.3) 4 (8.7) 4 AM E TE NA 1 (0.8) 1 (1.3) 0 (0.0) Totl 66 (53.2) 35 (44.9) 31 (67.4) 5 AM E CIP TE NA 4 (3.2) 2 (2.6) 2 (4.3) Totl 4 (3.2) 2 (2.6) 2 (4.3) NA=Nlidixic cid, TE=Tetrcycline, CIP=Ciprofloxcin, AM=Ampicillin, E=Erythromycin, GM=Gentmicin, C=Chlormphenicol we suggested tht it ws possible tht previous flocks contminted the tested flocks s reported by some uthors [18,20]. Giving dirty drinking wter could be nother risk fctor. As it contined fethers nd ws not chlorinted, we suggested tht Cmpylobcter cn be present s biofilms in wter s reported by some uthors [18]. Furthermore, chlorinted wter cn reduce the risk of broiler coloniztion [21,22], but it ws not the cse for our study. In frm A, the rte of thermophilic Cmpylobcter ws higher thn the rtes registered in frms B nd C. The presence of severl niml species ws more detected in frm A thn frms B nd C. They not only included dogs, cts, wild birds, nd rodents s in frms B nd C but lso wild bors. In ddition, rbbits nd bees were rered in the sme frm. Furthermore, the qulity of the litter could increse the rte of Cmpylobcter in frm A where the litter ws spent litter but moist in contrst to the other frms (66.7%) where litter ws fresh litter but dry. However, some uthors reported tht spent litter is more bctericidl thn fresh litter, but Cmpylobcter might be less ble to survive in dry litter [23]. Moreover, Szlnski et l. [24] isolted Cmpylobcter spp. from filth flies present in turkey housing for the 1 st time in the USA with contmintion rte of 23.3%. Thus, the existence of house flies in 66.7% of niml production fcilities (Frms A nd B) my contribute to turkey nd humn contmintion. Nevertheless, the lowest rte of contmintion ws found in frm B. Despite the presence of significnt sources of contmintion in this frm; subjects were rered within plstic greenhouse where only turkeys were rised in contrst to the other frms (A nd C) where turkeys were rered fter broilers. However, no deduction cn be done becuse no study hs reported if the prevlence of Cmpylobcter depends on the type of housing. In the visited slughterhouses, thermophilic Cmpylobcter ws isolted from ll the cecl content smples with very high prevlence rnging from 80 to 95% (Tble-2). However, lower rtes were observed in mny developed countries like the USA (55%) [9]. According to Jeffrey et l. [25], flock contmintion by thermophilic Cmpylobcter is most likely reflected by intestinl smples in slughterhouse; this suggests tht ll the frms which provided these positive flocks were highly contminted by thermophilic Cmpylobcter. Besides, the rering period tht seems decisive for intestinl coloniztion, crrige of Cmpylobcter spp. could be incresed by stressful events involved by trnsport nd non-complince of both feed withdrwl nd rest period tht were observed in ll the visited slughterhouses [26]. It seems estblished tht contmintion of crcsses during processing occurs directly through the intestinl contents or indirectly through equipment nd wter [27]. In trditionl slughterhouses nd modern slughterhouse, workers could lso be source of contmintion (Tble-1). In trditionl slughterhouses, A, B, nd C, thermophilic Cmpylobcter ws isolted from neck skin smples with n overll prevlence of 37.1%. In these estblishments, significnt points of cross-contmintion during processing represented by sclding, defethering, nd eviscertion [28] were bsent or done mnully. Besides, the bsence of sclding step in the visited trditionl slughterhouses (100.0%), defethering ws mnul, nd crcsses eviscertion ws performed on Veterinry World, EISSN: 2231-0916 1078
tble where the viscer were removed by the workers towrd the legs nd not towrd the hed. Therefore, crcsses contmintion could not be relted directly to the gut content of the sme subject, but it could be relted to fethers contmintion by fecl droppings. As mentioned bove, workers cn constitute n importnt source of trnsmission nd dissemintion of Cmpylobcter strins in the visited slughterhouses. However, initil contmintion of crcsses or initil source of fecl contmintion occurs either directly from gut content or indirectly notbly during sclding, defethering, or eviscertion step [28]. By observing the eviscertion method in trditionl slughterhouses, we concluded tht the first contmintion of crcsses ws cused by fecl contmintion of fethers probbly t frms, during trnsport or t slughterhouses [29]. Furthermore, to not contminte themselves first with intestinl contents, workers were very creful t the eviscertion step becuse, unfortuntely, they did not wer gloves. All the more so Jcobs-Rietsm hs reported tht fecl contmintion of fethers represents n importnt source of Cmpylobcter for poultry crcsses [30]. This observtion my explin the fct tht the prevlence of thermophilic Cmpylobcter ws higher in cecl contents thn in neck skins (p<0.05). A high prevlence (96.7%) of thermophilic Cmpylobcter ws noticed in modern slughterhouse D. In this processing plnt; neck skin could be contminted either indirectly or directly. However, vrious studies hve considered tht the sclding wter represents significnt source of contmintion [8,28], but, in our study, where turkey lot ws processed just fter processing broiler lot, the sclding step my hve prevented cross-contmintion between flocks. Indeed, scld temperture ws t 60 C, nd ccording to Snchez et l. [31], temperture superior to 53 C does not llow for the survivl of Cmpylobcter. Indirect contmintion could, however, be relted to the slughtering equipment notbly the fether removl mchine tht my enble recontmintion nd cross-contmintion between crcsses belonging to the sme flock [32]. Defethering opertion hs been reported s significnt source of cross-contmintion becuse the equipment led to intestinl content expulsion [4]. Furthermore, s reported by Berrng et l. [33], except direct contct with the skin, most of Cmpylobcter isoltes tht re crried into the equipment fter defethering re found on the skin. Direct contmintion of neck skins my be relted to gut contents which could increse the rte of thermophilic Cmpylobcter in the modern slughterhouse. According to Frnchin et l. [32] nd Gruntr et l. [34], during the eviscertion step, the intestinl rupture with extrvstions of its content is lwys possible, nd this stge represents the min fctor responsible for cross-contmintion tht cn led to substntil increse in Cmpylobcter detection during processing. It is recognized tht since the 20 th century, the number of Cmpylobcter strins isolted from humn smples resistnt to E nd/or CIP is incresing [35]. Similr rtes of ntibiotic resistnce to CIP (73.7%), E (21.1%) [36], chlormphenicol (0.0%), nd GM (0.0%) [37] were reported in USA nd Germny. Higher resistnce rtes thn our results were lso registered in the USA; 84.2% ws recorded to AM, 10.5% to chlormphenicol, nd 5.3% to GM [36]. Lower resistnce rtes thn ours were noticed in Germny regrding NA (65.3%), CIP (64.5%), nd TE (47.7%) [37]. In Algeri, quinolones, E, TE, nd AM re used for therpeutic purposes in poultry flocks. However, the use of chlormphenicol nd GM is prohibited [38]. These dt my explin the fct tht Cmpylobcter strins isolted from slughterhouses showed only resistnce to fmilies of ntibiotics tht re used in curtive tretment. The high rtes of resistnce nd multiresistnce, nd the frequent resistnce ptterns tht were reported could be relted not only to the uncontrolled dministrtion of some ntibiotics but lso to the extended use (16-20 weeks) of the tested ntibiotics in turkey frms. Durtion of the breeding period plys significnt role in incresing the number of resistnt Cmpylobcter strins to ntibiotics [39]. Comprison of ntimicrobil resistnce rtes of thermophilic Cmpylobcter strins isolted from cecl contents nd neck skins showed tht the difference between these results for ech tested ntibiotic ws not sttisticlly significnt (p>0.05). Rtes of resistnce to NA, E, CIP, TE, nd AM for Cmpylobcter strins tht were isolted from ech slughterhouse (A, B, C, nd D) were significntly different (p<0.05) (Tble-4). This deference could be relted to severl fctors such s the region nd the uncontrolled dministrtion of ntibiotics in flocks. Indeed, previous study concerning the ntimicrobil susceptibility of Slmonell isoltes in Algeri reported tht the use of different ntibiotics is widespred nd uncontrolled in poultry frms [40]. All the tested strins were resistnt to t lest one ntibiotic (100%), nd 96.9% of the isolted strins were multiresistnt (resistnce to t lest two ntibiotics). Our results re comprble to those reported by other uthors [36,41]. Resistnce to CIP nd/or E ws found in most of the isoltes tht were multidrug-resistnt. As described by D Lim et l. [41], the selection pressure generted by the use of different ntibiotics in turkey frms represents the cuse of the cquisition of vrious resistnce profiles. Furthermore, except for one drug resistnce pttern (AM-CIP), ll the ntimicrobil resistnce ptterns tht were observed in neck skin isoltes belong to drug resistnce ptterns of cecl content isoltes. This observtion suggests tht ll neck skin isoltes derived from cecl contents of the sme flock nd cn confirm one more time tht there ws no cross-contmintion between different flocks in the visited slughterhouses. We suggested Veterinry World, EISSN: 2231-0916 1079
this becuse s we found the sme ntimicrobil resistnce ptterns between neck skin nd cecl content isoltes (more resistnce ptterns for cecl content thn neck skin isoltes) (Tble-5), it ws possible tht cecl content isoltes of the sme flock contminted neck skins. If the results were different or if we found more resistnce ptterns for neck skin thn cecl content isoltes, then this could be relted to the presence of other strins from nother flock t the time of slughter. Furthermore, Peyrt et l. [42], by observing only resistnce rte results, found tht resistnce rtes of Cmpylobcter strin isolted from fecl droppings before slughter nd from neck skins fter slughter were the sme. They suggested in their study tht the slughtering process cnnot be considered s source of re-contmintion becuse it did not select strins resistnt to ntibiotics. Moreover, ccording to the slughtering process used in trditionl nd modern slughterhouses, contmintion of crcsses by other strins of previous flocks with other or new ntimicrobil resistnce ptterns seems impossible. Conclusion Our results reveled tht thermophilic Cmpylobcter ws isolted with high prevlence in ll the smpled turkey flocks where severl potentil sources of contmintion were found. Contmintion of turkey crcsses seemed to occur more in modern slughterhouse thn in trditionl slughterhouse where defethering nd eviscertion opertions represented significnt sources of contmintion. Most tested strins exhibited resistnce to E nd/or CIP. It is lrming becuse these ntibiotics re considered first-choice ntibiotics for humn cmpylobcteriosis. As turkey met production is incresing, humn cmpylobcteriosis cn increse too. For this reson, our results suggest tht the turkey industry in Algeri could be the cuse of mjor public helth problem through the spred of pthogenic strins of Cmpylobcter, s well s ntibiotic resistnce. It is more thn necessry to prevent Cmpylobcter contmintion in Algeri from frm to fork by estblishing preventive progrms like HACCP long ll the poultry production chin. Furthermore, the epidemiologicl surveillnce network of this foodborne pthogen should be estblished. Authors Contributions RB conceived, designed the study nd drfted the mnuscript under the supervision of TMH. RB nd SM designed the experiment protocol under the supervision of MN nd TMH. RB collected nd nlyzed smples. RB nd SZ did the sttisticl nlysis. RB nd LB revised the mnuscript under the supervision of MN nd TMH. All uthors red nd pproved the finl mnuscript. Acknowledgments We express our grtitude to Ministries of Defense nd Higher Eduction nd Scientific Reserch of Algeri for finncil support. The uthors re thnkful to the microbiology tem of Centrl Militry Hospitl of Algiers. Competing Interests The uthors declre tht they hve no competing interests. References 1. WHO. (2018) Cmpylobcter. Fcts Sheets. Genev, Switzerlnd. Avilble from: http://www.who.int/medicentre/fctsheets/fs255/en/. Accessed on 11-04-2018. 2. Berger, S. (2018) Cmpylobcteriosis: Globl Sttus. Gideon Series. 2018 th ed. The Seed Time of the Republic, New York. p141. 3. Dromigny, E. (2007) In: Lvoisier, T.C., editor. Monogrphie de Microbiologie: Cmpylobcter. 1 st ed. Frnce, Pris. p283. 4. Logue, C.M., Sherwood, J.S., Elijh, L.M., Olh, P.A. nd Dockter, M.R. (2003) The incidence of Cmpylobcter spp. on processed turkey from processing plnts in the Midwestern United Sttes. J. Appl. Microbiol., 95: 234-241. 5. Szczepnsk, B., Andrzejewsk, M., Spic, D. nd Klwe, J.J. (2017) Prevlence nd ntimicrobil resistnce of Cmpylobcter jejuni nd Cmpylobcter coli isolted from children nd environmentl sources in Urbn nd Suburbn res. BMC Microbiol., 17(80): 1-9. 6. Zhou, J., Zhng, M., Yng, W., Fng, Y., Wng, G. nd Hou, F. (2016) A seventeen-yer observtion of the ntimicrobil susceptibility of clinicl Cmpylobcter jejuni nd the moleculr mechnisms of erythromycin-resistnt isoltes in Beijing, Chin. Int. J. Infect. Dis., 42: 28-33. 7. Mégrud, F., Boudr, G., Bessoud, K., Bensid, S., Dbis, F., Soltn, R. nd Touhmi, M. (1990) Incidence of Cmpylobcter infection in infnts in western Algeri nd the possible protective role of brestfeeding. Epidemiol. Infect., 105: 73-78. 8. Rhimi, E., Momtz, H. nd Bonydin, M. (2010) PCR detection of Cmpylobcter sp. from turkey crcsses during processing plnt in Irn. Food Contr., 21: 692-694. 9. Kshom, I.P.B., Kumr, A., Snd, Y.M., Gebreyes, W., Kzwl, R.R., Grbed, R. nd Rjshekr, G. (2014) Phenotypic nd genotypic diversity of thermophilic Cmpylobcter spp. in commercil turkey flocks: A longitudinl study. Foodborne Pthog. Dis., 11(11): 850-860. 10. Ingres-Cpccioni, S., Gonz lez-bod ı, S., Jim enez- Trigos, E., Mrco-Jim enez, F., Ctl, P., Veg, S. nd Mrin, C. (2015) Comprison of different smpling types cross the rering period in broiler flocks for isoltion of Cmpylobcter spp. Poult. Sci., 94: 766-771. 11. OIE. (2004) Cmpylobcter jejuni nd Cmpylobcter coli. OIE Mnul of Dignostic Tests nd Vccines for Terrestril Animls. Chpter 2.10.8, Genev: Switzerlnd. p1177-1187. 12. OIE. (2017) Infection with Cmpylobcter jejuni nd Cmpylobcter coli. OIE Mnul of Dignostic Tests nd Vccines for Terrestril Animls. Chpter 2.9.3, Genev: Switzerlnd. p1-9. 13. Sndberg, M., Østensvik, Ø., Aunsmo, A.L., Skjerve, E. nd Hofshgen, M. (2006) An evlution of smpling nd culturing methods in the Norwegin ction pln ginst Cmpylobcter in broilers. Int. J. Food Microbiol., 106(3): 313-317. 14. WHO. (2003) Isolement, Identifiction et Détermintion de l Sensibilité Aux Antibiotiques des Cmpylobcter. Globl Slm-Surv. 4 th ed. Psteur Interntionl, Frnce. p1-30. 15. ISO 10272. (1995) Microbiologie des Aliments - Méthode Horizontle Pour l Recherche des Cmpylobcter Thermotolérnts. 1 st ed. AFNOR, Frnce. p1-15. 16. CA-SFM. (2010) Comité de L ntibiogrmme de l Veterinry World, EISSN: 2231-0916 1080
Société Frnçise de Microbiologie. 2010 th ed. Avilble from: http://www.sfm-microbiologie.org/userfiles/files/ csfm_2010.pdf. Accessed on 25-06-2010. 17. Prkr, S.F.D., Schdev, D., desouz, N., Kmble, A., Suresh, G., Munot, H., Hngl, D., Shouche, Y. nd Kpdnis, B. (2013) Prevlence, sesonlity nd ntibiotic susceptibility of thermophilic Cmpylobcters in cec nd crcsses of poultry birds in the live-bird mrket. Afr. J. Microbiol. Res., 7(21): 2442-2453. 18. Robyn, J., Rsschert, G., Psmns, F. nd Heyndrickx, M. (2015) Thermophilic Cmpylobcter during broiler rering: Risk fctors nd Intervention. Compr. Rev. Food Sci. Food Sf., 14: 81-105. 19. Cox, N.A., Stern, N.J., Crven, S.E., Berrng, M.E. nd Musgrove, M.T. (2000) Prevlence of Cmpylobcter nd Slmonell in the cecl droppings of turkeys during production. J. Appl. Poult. Res., 9: 542-545. 20. Arsenult, J., Letellier, A., Quessy, S., Normnd, V. nd Boulinne, M. (2007) Prevlence nd risk fctors for Slmonell spp. And Cmpylobcter spp. cecl coloniztion in broiler chicken nd turkey flocks slughtered in Quebec, Cnd. Prev. Vet. Med., 81(4): 250-64. 21. Person, A.D., Greenwood, M.H., Heling, T.D., Rollins, D., Shhmt, M., Donldson, J. nd Colwell, R.R. (1993) Coloniztion of broiler chickens by wterborne Cmpylobcter jejuni. Appl. Environ. Microbiol., 59: 987-996. 22. Ellis-Iversen, J., Jorgensen, F., Bull, S., Powell, L., Cook, A.J. nd Humphrey, T.J. (2009) Risk fctors for Cmpylobcter colonistion during rering of broiler flocks in Gret Britin. Prev. Vet. Med., 89: 17884. 23. Line, J.E. (2006) Influence of reltive humidity on trnsmission of Cmpylobcter jejuni in broiler chickens. Poult. Sci., 85: 1145-1150. 24. Szlnski, A.L., Owens, C.B., Mcky, T. nd Steelmn, C.D. (2004) Detection of Cmpylobcter nd Escherichi coli O157:H7 from filth flies by polymerse chin rection. Med. Vet. Entomol., 18: 241-246. 25. Jeffrey, J.S., Tonook, K.H. nd Lozno, J. (2001) Prevlence of Cmpylobcter spp. from skin, crop, nd intestine of commercil broiler chicken crcsses t processing. Poult. Sci., 80: 1390-1392. 26. Wesley, I.V., Murok, W.T., Trmpel, D.W. nd Hurd, H.S. (2005) Effect of preslughter events on prevlence of Cmpylobcter jejuni nd Cmpylobcter coli in mrket-weight turkeys. Appl. Environ. Microbiol., 71: 2824-2831. 27. Zendehbd, B., Arin, A.A. nd Alipour, A. (2013) Identifiction nd ntimicrobil resistnce of Cmpylobcter species isolted from poultry met in Khorsn province, Irn. Food Contr., 32(2): 724-727. 28. Shne, S.M. (1992) The significnce of Cmpylobcter jejuni infection in poultry: A review. Avin. Pthol., 21: 189-213. 29. Buhr, R.J., Berrng, M.E. nd Cson, J.A. (2003) Brest skin: Bcteril recovery from brest skin of geneticlly fethered nd fetherless broiler crcsses immeditely following sclding nd picking. Poult. Sci., 82(10): 1641-1647. ******** 30. Jcobs-Rietsm, W. (2000) Cmpylobcter in the food supply. In: Nchmkin, I. nd Blser, M.J., editors. Cmpylobcter. 2 nd ed. Americn Society of Microbiology Press, Wshington, DC. p467-482. 31. Snchez, M.X., Fluckey, W.M., Brshers, M.M. nd McKee, S.R. (2002) Microbil profile nd ntibiotic susceptibility of Cmpylobcter spp. nd Slmonell spp. in broilers processed in ir-chilled nd immersion-chilled environments. J. Food Protect., 65: 948-956. 32. Frnchin, P.R., Oglir, P.J. nd Btist, C.R.V. (2007) Frequency of thermophilic Cmpylobcter in broiler chickens during industril processing in Southern Brzil. Poult. Sci., 48: 127-132. 33. Berrng, M.E., Buhr, R.J., Cson, J.A. nd Dickens, J.A. (2002) Microbiologicl consequences of skin removl prior to eviscertion of broiler crcsses. Poult. Sci., 81: 134-138. 34. Gruntr, I., Bisizzo, M., Kušr, D., Pte, M. nd Ocepek, M. (2015) Cmpylobcter jejuni contmintion of broiler crcsses: Popultion dynmics nd genetic profiles t slughterhouse level. Food Microbiol., 50: 97-101. 35. Sierr-Arguello, Y.M., Perdoncini, G., Morgn, R.B., Slle, C.T.P., Mores, H.L.S., Gomes, M.J.P. nd Nscimento, V.P. (2016) Fluoroquinolone nd mcrolide resistnce in Cmpylobcter jejuni isolted from broiler slughterhouses in southern Brzil. Avin Pthol., 45(1): 66-72. 36. Noormohmed, A. nd Fkhr, M.K. (2014) Prevlence nd ntimicrobil susceptibility of Cmpylobcter spp. in Oklhom conventionl nd orgnic retil poultry. Open. Microbiol. J., 8: 130-137. 37. El-Adwy, H., Ahmed, M.F., Hotzel, H., Tomso, H., Tenhgen, B.A, Hrtung, J., Neubuer, H. nd Hfez, H.M. (2014) Antimicrobil susceptibilities of Cmpylobcter jejuni nd Cmpylobcter coli recovered from orgnic Turkey frms in Germny. Poult. Sci., 94(11): 2831-2837. 38. MADRP-DSV. (2006) Ministry of Agriculture, Rurl Development nd Fisheries-Veterinry Services Deprtment. Authoristion nd supervision of medicinl products for veterinry use. Decision NO. 644. p1-6. 39. Nyk, R., Stewrt, T., Nwz, M. nd Cernigli, C. (2006) In vitro ntimicrobil susceptibility, genetic diversity nd prevlence of UDP-glucose 4-epimerse (gle) gene in Cmpylobcter coli nd Cmpylobcter jejuni from Turkey production fcilities. Food Microbiol., 23: 379-392. 40. Mezli, L. nd Hmdi, T.M. (2012) Prevlence nd ntimicrobil resistnce of Slmonell isolted from met nd met products in Algiers (Algeri). Foodborne Pthog. Dis., 9(6): 522-529. 41. D lim, C.B., Miller, W.G., Mndrell, R.E., Wright, S.L., Siletzky, R.M., Crver, D.K. nd Kthriou, S. (2007) Clonl popultion structure nd specific genotypes of multidrug-resistnt Cmpylobcter coli from turkeys. Appl. Environ. Microbiol., 73: 2156-2164. 42. Peyrt, M.B. (2008) Étude de L influence du Nettoyge et de l Désinfection et des Procédés D bttge en Abttoir de Volilles sur le Niveu de Résistnce Aux Antibiotiques des Cmpylobcters. Thèse de Doctort. Université de Rennes 1. p237. Veterinry World, EISSN: 2231-0916 1081