Shng et l. BMC Veterinry Reserch (2018) 14:257 https://doi.org/10.1186/s12917-018-1590-1 RESEARCH ARTICLE Open Access Distribution nd dissemintion of ntimicrobil-resistnt Slmonell in broiler frms with or without enrofloxcin use Ke Shng, Bi Wei nd Min Kng * Abstrct Bckground: Slmonell is mjor zoonotic food-borne pthogen tht persists on poultry frms, nd nimls undergo reinfection with endemic strins. The present study imed to investigte the chrcteristics nd dissemintion of ntimicrobil-resistnt Slmonell within nd between broiler frms tht used enrofloxcin nd those tht did not. Results: Clocl nd environmentl (litter, feed, nd wter) smples from two selected flocks in ech of 12 frms owned by the sme compny were collected three times over 30-dy period of two production cycles during 2015 2016. The rte of Slmonell isoltion ws 7.8% (123/1584). Nine Slmonell serotypes (116 isoltes) nd seven untypble isoltes were identified, nd Slmonell Montevideo ws the most prevlent serotype. Azithromycinresistnt (17.9%) nd colistin-resistnt (3.3%) isoltes were detected, nd multidrug-resistnt isoltes (43.1%) were lso observed. No isolte ws resistnt to enrofloxcin or ciprofloxcin; however, intermedite resistnce to enrofloxcin ws significntly higher (P < 0.05) in frms tht used enrofloxcin thn in those tht did not. The rte of multi-drug resistnce mong litter isoltes (25/44, 56.8%) ws significntly higher (P < 0.05) thn tht mong clocl swb (24/67, 35.8%) nd feed (4/12, 33.3%) isoltes. Pulsed-field gel electrophoresis (PFGE) nlysis of strins of the sme serotype ws conducted to determine their epidemiologicl reltionship. The PFGE types were clssified into 31 groups with 100% correltion cutoff in dendrogrms for Slmonell Montevideo isoltes, which showed 100% genomic identity bsed on ge, smple type, flock, nd production cycle within nd between frms. Conclusion: The present study highlights the occurrence of horizontl trnsmission nd cyclic contmintion with ntimicrobil-resistnt Slmonell in broiler frms owned by the sme compny. Litter my be good indictor of indoor environmentl contmintion with ntimicrobil-resistnt Slmonell on frms. Additionlly, enrofloxcin use my be one of the fctors promoting resistnce towrds it in Slmonell. Keywords: Slmonell, Antimicrobil resistnce, Circulting contmintion, Enrofloxcin, Broiler frm, Litter, PFGE Bckground Slmonell is the leding cuse of food-borne illness worldwide, nd dissemintion of ntimicrobil-resistnt Slmonell through the food chin, especilly through chicken, hs importnt implictions for the filure of slmonellosis tretment. There is incresing risk of chicken-medited spred of ntimicrobil-resistnt Slmonell to public helth [1]. The prevlence of * Correspondence: vet.minkng@gmil.com Ke Shng nd Bi Wei contributed eqully to this work. Deprtment of Veterinry Infectious Diseses nd Avin Diseses, College of Veterinry Medicine nd Center for Poultry Diseses Control, Chonbuk Ntionl University, 79 Gobong-ro, Iksn 54596, South Kore Slmonell on frms is linked to the prevlence of Slmonell in the derived met products [2]. Prevention of Slmonell contmintion in chicken products requires detiled knowledge of the mjor sources of contmintion. Although mesures for eliminting Slmonell on breeder frms through vccintion, use of ll-in/ll-out replcement systems on broiler frms, nd ntimicrobil-free strtegies hve been implemented, high Slmonell prevlence rtes nd ntimicrobil-resistnce rtes re still observed in broiler frms [3, 4]. Interventions performed t poultry frms, including enhnced biosecurity, rodent control, routine surveillnce of the frm environment, feed decontmintion, nd use of The Author(s). 2018 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.
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 2 of 14 utogenous poultry vccine, cn reduce, but cnnot eliminte Slmonell from live poultry [5]. Environmentl smples, especilly poultry litter, hve been reported to be good indictor for the presence of Slmonell in poultry frms [6, 7]. We speculted tht the trnsmission of ntimicrobil-resistnt Slmonell mong frms might occur through indoor environmentl contmintion in frms. Slmonell strins cn develop ntimicrobil resistnce (AMR), initilly to the trditionl first-line drugs chlormphenicol, mpicillin, nd trimethoprim/sulfmethoxzole. Becuse of such resistnce, fluoroquinolones (FQs), third-genertion cephlosporins, nd mcrolides (zithromycin) hve become criticlly importnt for treting slmonellosis in humns [8]. Prophylctic tretment of poultry with enrofloxcin (ENR), fluoroquinolone ntibiotic used to tret niml infections, hs been implicted in the incresing resistnce to ciprofloxcin, posing risk to humn helth [9 11]. In Kore, ENR ws licensed for veterinry use in 1987, nd the quntity of ENR used hs incresed since its introduction [12]. In fct, the quntity of ENR sold ws the highest of ll ntimicrobils used to tret chicken in Kore [13]. Concerns over the entry of ntimicrobil-resistnt zoonotic bcteri into the food chin nd the consequent humn infections led the Food nd Drug Administrtion (FDA) to bn the use of ENR in poultry in the USA in September 2005 [14]. The use of ntimicrobils in poultry frming nd the ccompnying selection pressure for resistnt Slmonell hve been the subjects of numerous studies [15, 16]. Antimicrobil use in frming hs led to the widespred dissemintion of ntimicrobil-resistnt Slmonell in broiler frms [4]. Therefore, to institute effective mesures for reducing the infection of chicken with ntimicrobil-resistnt Slmonell, Slmonell contmintion on broiler frms needs to be well-understood. To explore production cycle-contmintion with ntimicrobil-resistnt Slmonell in broiler frms tht use ntimicrobils nd in those tht do not, smples were collected from 12 frms (which either used or did not use ENR) owned by single compny during two production cycles. Trce-bck investigtions of the reltionship between ntimicrobil-resistnt Slmonell nd ENR use in broiler frms hve not been previously conducted. The present study ws imed t determining the distribution nd spred of indoor contmintion with ntimicrobil-resistnt Slmonell through comprehensive investigtion of its prevlence within nd between broiler frms. We lso determined the epidemiologicl reltionships mong strins of the sme serotype using electrophoresis -bsed DNA fingerprinting. Methods Smpling The smples used in the present study were collected specificlly for the purposes of the study. In totl, 1584 fresh smples including 660 clocl smples nd 924 environmentl smples (396 litter smples, 264 feed smples, nd 264 wter smples), were collected during 2015 nd 2016 from 12 frms belonging to one of the lrgest integrted poultry compnies in South Kore. In seven frms, ENR (50 mg/l) hs been dministered to young chicks vi ddition to drinking wter for three consecutive dys, while on the other five frms; no ENR ws used [17]. The sme ENR dministrtion protocol ws followed in ll seven frms. The smpling frms contined n verge of 70 100 thousnds broilers, nd 3 5 seprte flocks. Two flocks per frm were smpled three times over thirty-dy period (chickens t 1, 15, nd 30 dys of ge) during one production cycle. This smpling ws then repeted during second, seprte production cycle. The clocl smples were rndomly collected from 25 broilers in entire re of the flock, nd smples from five broilers were pooled into one test smple. The environmentl smples, including litter (n = 15), feed (n = 10), nd wter (n = 10) were uniformly collected from equl res of the flock, nd five smples obtined from sme re were pooled into one test smple. Finlly clocl swbs (S, n = 5), litter (L, n = 3), feed (F, n = 2) nd wter (W, n = 2) were collected from ech flock. Slmonell isoltion Smples were collected in sterile plstic conicl tubes (50 ml; SPL Life Sciences Co., Ltd., Seoul, Kore) nd were stored under refrigertion in the lbortory until nlysis, which ws performed within 48 h of rrivl. Fresh smples [1 g (or ml)] were seprtely mixed with 9 ml (1:9 dilutions) of buffered peptone wter (BPW; BD Difco, Sprks, MD, USA) nd incubted t 37 C for 24 h for enrichment. An liquot of the enriched BPW culture (100 μl) ws trnsferred to 10 ml of Rppport Vssilidis (RV) broth (Thermo Fisher Scientific, Oxoid Ltd., Bsingstoke, UK), nd incubted t 42 C for 24 h for selective enrichment [18]. A loopful of ech RV culture ws streked onto xylose-lysine-deoxycholte (XLD) gr plte (BD Difco XLD gr, USA), nd the plte ws incubted overnight t 37 C. Presumptive Slmonell colonies were then tested with Slmonell ltex test kit (Thermo Fisher Scientific, Oxoid Ltd., Bsingstoke, UK). Slmonell serotyping The serogroup nd serovr of ech Slmonell isolte were identified following Edwrds nd Ewing s procedure for the identifiction of Enterobctericee using n
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 3 of 14 ntiser kit (BD Difco, Sprks, MD, USA) [19]. Somtic O ntigen (BD Difco) ws identified using the slide gglutintion test with commercilly vilble ntiserum. Flgellr (H) ntigens (phses 1 nd 2) were identified vi successive inocultion onto 0.3% brin hert infusion (BHI) gr (BD) to ctivte flgell, followed by inocultion into BHI broth. The broth ws cultured overnight, fixed with 0.6% formlin, nd then nlyzed using tube gglutintion test [20]. Antimicrobil susceptibility The minimum inhibitory concentrtions (MICs) of the test ntimicrobils nlidixic cid (NAL), ciprofloxcin (CIP), neomycin (NEO), gentmicin (GEN), streptomycin (STR), tetrcycline (TET), zithromycin (AZM), moxicillin/clvulnic cid (AMC), cefoxitin (FOX), ceftiofur (XNL), mpicillin (AMP), trimethoprim/sulfmethoxzole (SXT), colistin (COL), florfenicol (FFN), nd chlormphenicol (CHL) were determined using the Sensititre pnel KRNV4F (TREK Dignostic Systems, Kore), while the MICs of enrofloxcin (ENR), tigecycline (TIG), nd fosfomycin (FOS) were determined using the gr dilution method. Escherichi coli (ATCC 25922) were used s the qulity-control strin. The susceptibility brekpoints of most tested ntimicrobils were interpreted bsed on CLSI guidelines [21], while those of XNL, ENR, nd FFN were interpreted bsed on CLSI BM31-A3 stndrds [22]. No CLSI interprettion criteri were vilble for STR, TIG, COL, nd NEO; therefore, the following MIC vlues were considered to indicte resistnce: STR, 64 μg/ml [23]; TIG, 8 μg/ml [24]; COL, 4 μg/ml [25]; nd NEO, 16 μg/ml [26] (Tble 1). Slmonell isoltes resistnt to t lest three ntimicrobil clsses were designted multidrug resistnt (MDR). PFGE nd BioNumerics nlysis Isoltes of Slmonell Montevideo (n = 75) nd Slmonell Senftenberg (n = 16) were genotyped using PFGE following protocols of the Centers for Disese Control nd Prevention vilble on PulseNet, with some modifictions. In brief, the Slmonell isoltes were streked onto McConkey gr pltes nd incubted overnight t 37 C. Then, the bcteri were suspended in PBS t n OD vlue of 0.6 0.8. Genomic DNA (extrction using 1% SDS nd 1 mg/ml proteinse K, Biosesng, Seoul, Kore) smples were digested with 50 U of XbI (Thermo Fisher Scientific, Inchon, Kore) t 37 C for 3 h. The digested DNA ws seprted by electrophoresis in 0.5 x TBE buffer t 14 C for 18 h using CHEF-DR @ electrophoresis system (Bio-Rd, Hercules, CA, USA). The pulse time ws rmped from 2.16 to 63.8 s. Tble 1 Antimicrobils used in the study nd the tested concentrtion rnges Antimicrobils Abbrevition Brekpoints (μg/ml) Concentrtion rnges S b I R (μg/ml) Nlidixic cid NAL 16 - c 32 2 128 Ciprofloxcin CIP 0.06 0.12 0.5 1 0.12 16 Enrofloxcin ENR 0.25 0.5 1 2 0.12 64 Neomycin NEO 16 2 32 Gentmicin GEN 4 8 16 1 64 Streptomycin STR 32 64 2 128 Tetrcycline TET 4 8 16 2 128 Azithromycin AZM 16 32 0.25 64 Amoxicillin/clvulnic cid AMC 8/4 16/8 32/16 2/1 64/32 Cefoxitin FOX 8 16 32 1 32 Ceftiofur XNL 2 4 8 0.5 8 Ampicillin AMP 8 16 32 2 32 Trimethoprim/sulfmethoxzole SXT 2/38 4/76 0.12/2.38 4/76 Colistin COL 2 4 2 32 Florfenicol FFN 32 2 64 Chlormphenicol CHL 8 16 32 2 64 Tigecycline TIG 2 4 8 0.12 32 Fosfomycin FOS 64 128 256 0.25 256 Sorted bsed on ctegory of ntimicrobils b S, sensitivity; I, intermedite resistnce; R, resistnce c -, no stndrd brekpoint vlue in relted references
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 4 of 14 Slmonell Brenderup H9812, which ws included s moleculr weight stndrd, ws processed with ech btch of isolte. The gels were stined with ethidium bromide, nd the DNA ptterns were visulized on UV trnsillumintor (Bio Doc-It Imging System, Uplnd, CA, USA). The DNA fingerprints obtined with PFGE were nlyzed using BioNumerics (version 5.10 for Windows). Dice similrity coefficients were clculted bsed on pirwise comprison of the PFGE types of the isoltes. The isoltes were considered to hve closely relted bnd ptterns bsed on moleculr typing when their PFGE types hd dice similrity coefficients of 100%, nd were clustered t the 90% similrity level. Bnd-mtching settings, with n optimiztion of 1.0% nd position tolernce of 1.0%, were pplied. Sttisticl nlysis The chi-squre test ws used to test for significnt differences in the rtes of Slmonell isoltion, MDR prevlence, nd AMR rtes between frms tht used ENR nd those tht did not. P vlues less thn 0.05 were considered sttisticlly significnt. Anlyses were performed using SPSS version 19.0 (IBM Co., Armonk, NY, USA). Results Prevlence nd serovrs of Slmonell The clocl nd environmentl smples (n = 1584) collected from the broiler frms were nlyzed for the presence of Slmonell. In totl, 123 (7.8%) smples were positive for Slmonell. There were no significnt differences in isoltion rtes between the litter smples (44/ 396, 11.1%) nd the clocl swbs (67/660, 10.2%); however, significntly higher isoltion rtes were found in litter smples nd clocl swbs compred to feed (12/264, 4.5%), nd wter smples (0/264, 0.0%) (Fig. 1). The Slmonell isoltion rte from ll types of smples ws significntly lower (P < 0.05) in frms tht used ENR (55/924, 6.0%) thn on frms tht did not (68/660, 10.3%; Tble 2). Isoltes were ssigned to nine serovrs, most of which belonged to serogroups C1 (65.0%) nd E1 (17.1%). S. Montevideo (75 isoltes, 61.0%) ws the dominnt serovr, nd S. Senftenberg (21 isoltes, 17.1%) ws distnt second, followed by S. Emek (9 isoltes, 7.3%), S. Mcclesfield (4 isolte, 3.3%), nd S. Virchow (3 isoltes, 2.4%). Only one isolte ws detected for the four serotypes, S. Infntis, S. Edinburg, S. Hto, nd S. Vellore, nd seven isoltes (5.7%) mrked s S. spp. could not be ssigned to specific serotypes (Tble 3). A C Isoltion rte 12.0 10.0 rte Antimicrobils-resistnce 8.0 6.0 4.0 2.0 0.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Clocl swb Litter Feed Wter b b c B 50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 Clocl swb Litter ENR use (n = 55) No ENR use (n = 68) NAL ENR (I) CIP (I) NEO STR TET AZM FOX SXT FFN AMP XNL COL Fig. 1 Rtes of Slmonell isoltion from smples of clocl swbs, litter, feed, nd wter in broiler frms (); the incidence of multi-drug resistnce in isoltes from clocl swbs, litter, feed, nd wter in broiler frms (b); rtes of ntimicrobil-resistnce in isoltes from frms tht used ENR nd in isoltes from frms tht did not (c). The chi-squre test ws used to ssess the significnce of differences. P vlues less thn 0.05 were considered sttisticlly significnt, nd were mrked with lowercse letters (/b/c) b drug resistnce rte Multi- b b Feed b
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 5 of 14 Tble 2 Slmonell isoltion from different smple types in broiler frms with or without enrofloxcin (ENR) use * ENR Smple % (no. positive/totl no. smples) use no. Clocl swb Litter Feed Wter Totl Yes 924 7.5 (29/385) 8.2 (19/231) 4.5 (7/154) 0.0 (0/154) 6.0 (55/924) No 660 13.8 (38/275) b 15.2 (25/165) b 4.5 (5/110) 0.0 (0/110) 10.3 (68/660) b Totl 1584 10.2 (67/660) 11.1 (44/396) 4.5 (12/264) 0.0 (0/264) 7.8 (123/1584) * Lowercse (/b) were used to indicte significnt difference in isoltion rtes between frms with enrofloxcin use nd those without; different letters indicte significnt differences (P < 0.05) Antimicrobil susceptibility nlysis All the isoltes nlyzed in this study showed resistnce to t lest one tested ntimicrobil (Tble 3). Resistnce to NAL ws the most prevlent (120/123, 97.6%), followed by tht to NEO (62/123, 50.4%), STR (51/123, 41.5%), TET (28/123, 22.8%), AZM (22/123, 17.9%), FOX (17/123, 13.8%), XNL (5/123, 4.1%), AMP (3/123, 2.4%), SXT (9/123, 7.3%), COL (4/123, 3.3%), nd FFN (3/123, 2.4%). severl isoltes showed intermedite resistnce (IR) to ENR (46/123, 37.4%) nd CIP (42/123, 31.1%). All isoltes were sensitive to the five ntimicrobils CHL, GEN, TIG, FOS, nd AMC. Thirty-one AMR phenotypes were observed mong the Slmonell isoltes from the 12 broiler frms; the AMR profile NAL + NEO + STR + TET ws the most prevlent in this study (10 isoltes, 8.1%) (Tble 4). Further, seven MDR isoltes comprising four S. Montevideo, two S. spp., nd one S. Infntis isoltes, were resistnt to 5 ntimicrobil clsses, ll of which were isolted from the frms tht used ENR. The MDR rte mong litter isoltes (25/44, 56.8%) ws significntly higher (P < 0.05) thn tht mong the clocl swb (24/67, 35.8%) nd feed (4/12, 33.3%) isoltes (Fig. 1b). The rtio of IR to ENR ws significntly higher (P < 0.05) in isoltes from frms tht used ENR (26/55, 47.3%) thn in those from frms tht did not (20/68, 29.4%; Fig. 1c). Serotype distribution nd genetic nlysis of isoltes from broiler frms Serotype diversity ws observed in the broiler frms smpled in the present study with 75% (9/12) of them showing contmintion with t lest two Slmonell serovrs (Tble 5). S. Montevideo nd S. Senftenberg were isolted from 11 nd 8 of the 12 frms, respectively. In frms B, H, I, nd J, there were t lest four different serotypes observed in single flock. In one frm (frm H), isoltes from the sme flock t different ges hd the sme PFGE type (type 13), with similrity index of 100% (isoltes A15-CF-002-1S-3 nd A15-CF-003-1 L-1); isoltes from different flocks were of the sme type (type 13; isoltes A15-CF-003-1 L-1 nd A15-CF-003-2S-2); nd two isoltes, A15-CF-002-2 L-2 nd A15-CF-063-1 L-2, which were of the sme PFGE type (type 11) nd sme AMR profile (including intermedite ENR resistnce), were obtined from litter during different production cycles. Isoltes of the sme PFGE type were lso observed in different frms (type 5 in frms H, F, I, K, nd E; type 8 in frms B, E, D, J, C, F, nd A; type 11 in frms A, H, nd J; type 13 in frms B, E, H, nd J; type 17 in frms G nd J; type 22 in frms K nd E; nd type 26 in frms B nd K; Fig. 2). In S. Senftenberg isoltes, the sme PFGE type ws observed in two different frms in different cities, tht neither of which used ENR (type 4 in frms H nd J; type 6 in frms J nd K; Additionl file 1). Discussion In the present study, to explore the distribution nd dissemintion of ntimicrobil-resistnt Slmonell in broiler frms, we obtined smples during two production cycles from frms tht used ntimicrobils nd those tht did not (Tble 5). S. Montevideo, one of the serotypes prevlent in humn infections [27], showed high resistnce to 3 ntimicrobils (49.3%; Tble 3). In recent yers, there hs been n increse in S. Montevideo humn infections nd outbreks worldwide, including in the USA, Europe, Austrli, nd Asin ntions, such s South Kore [5, 28 30]. In the USA, the number of humn illnesses cused by S. Montevideo incresed from 728 cses in 2002 to 1203 cses in 2012 [31]. In South Kore, S. Montevideo is one of the most common serotypes ffecting the poultry industry [32, 33]. Additionlly, the most serious cse of resistnce found in the present study ws n S. Montevideo isolte with resistnce to eight ntimicrobils (NAL + NEO + STR + TET + FOX + XNL + AMP + AZM; Tble 4). In ddition, there ws n MDR S. Infntis isolte resistnt to 5 ntimicrobil clsses (Tble 3); nd S. Infntis is one of the most prevlent serotypes in South Kore nd second in frequency mong serovrs [34]. In countries including Germny [35], Hungry [36], Itly [37], nd Jpn [38], emergence nd clonl dissemintion of MDR S. Infntis strins in chicken hs been recent reported, nd hs been relted to n incresed incidence of humn infections [31]. Given these findings, further investigtions of the ntimicrobil resistnce nd distribution of S. Infntis in slughterhouses nd retil chicken met re required.
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 6 of 14 Tble 3 Antimicrobil resistnce in Slmonell isoltes from broiler frms Serovr (Serogroup) N (%) NAL NEO STR TET AZM XNL FOX AMP SXT COL FFN > 1 gent MIC 3 gents 5 clsses n (%) n (%) n (%) n (%) Hto (B) 1 (0.8) 1 1 1 0 0 0 0 0 0 0 0 1 1 0 Vellore (B) 1 (0.8) 0 1 0 0 0 0 0 0 0 0 0 1 0 0 Montevideo (C1) 75 (61.0) 128/128 75 4/32 35 (46.7) 32/ 128 37 (49.3) 4/16 24 (32) 16/32 12 (16) 1/1 3 (4.0) 4/16 9 (12.0) 4/8 2 (2.7) 0.5/1 8 (10.7) 2/2 2 (2.7) 8/8 3 (4.0) 75 37 (49.3) 4 (5.3) Virchow (C1) 3 (2.4) 128/128 3 16/32 3 64/64 2 (66.7) 8/8 0 64/64 2 (66.7) 1/1 0 16/32 1 (33.3) 8/8 0 0.25/0.25 0 2/2 0 8/8 0 3 2 (66.7) 0 Infntis (C1) 1 (0.8) 1 1 1 1 1 1 0 0 0 1 0 1 1 1 Edinburg (C1) 1 (0.8) 1 0 0 0 0 0 1 0 0 0 0 1 0 0 Emek (C2-C3) 9 (7.3) 128/128 9 4/16 3 (33.3) 8/32 1 (11.1) 1/4 0 16/32 3 (33.3) 0.5/1 0 4/16 0 2/4 0 0.5/0.5 0 2/2 0 4/8 0 9 3 (33.3) 0 Mcclesfield (D2) 4 (3.3) 64/128 4 2/16 1 (25) 8/128 1 (25) 1/16 1 (6.3) 16/64 1 (25) 0.5/1 0 4/32 0 2/8 0 0.5/4 1 (25) 2/2 0 2/8 0 4 1 (25) 0 Senftenberg (E4) 21 (17.1) 128/128 21 4/32 10 (47.6) 32/ 128 4 (19) 1/4 0 16/16 2 (9.5) 0.5/1 0 4/16 2 (9.5) 2/4 0 0.5/0.5 0 2/2 0 4/8 0 21 2 (9.5) 0 spp. (C2-C3, B, D1) 7 (5.7) 6 (71.4) 7 4 (57.1) 2 (28.6) 1 (14.3) 1 (14.3) 4 (57.1) 1 (14.3) 0 1 (14.3) 0 7 4 (57.1) 2 (28.6) Totl 123 128/128 120 (97.6) 8/32 62 (50.4) 8/32 51 (41.5) 1/16 28 (22.8) 16/32 22 (17.9) 1/1 5 (4.1) 4/32 17 (13.8) 4/8 3 (2.4) 0.5/0.5 9 (7.3) 2/2 4 (3.3) 8/8 3 (2.4) 123 53 (43.1) 7 (5.7) -, not nlyzed; the unit of MIC ws μg/ml
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 7 of 14 Tble 4 Antimicrobil resistnce profiles of Slmonell isoltes from broiler frms (n = 123) No. Antimicrobil resistnce profile n (%) Serovrs (n) Susceptible 0 1 NAL 27 (22.0) Montevideo (13), Senftenberg (8), Emek (5), Mcclesfield (1) 2 NEO 2 (1.6) Vellore (1), spp. (1) 3 NAL + NEO 18 (14.6) Montevideo (9), Senftenberg (6), Virchow (1), spp. (2) 4 NAL + STR 6 (4.9) Montevideo (5), Senftenberg (1) 5 NAL + AZM 4 (3.3) Montevideo (1), Senftenberg (1), Emek (1), Mcclesfield (1) 6 NAL + FOX 3 (2.4) Montevideo (1), Senftenberg (1), Edinburg (1) 7 NAL + SXT 9 (7.3) Montevideo (8), Mcclesfield (1) 8 NAL + COL 1 (0.8) Montevideo (1) 9 NAL + NEO + STR 7 (5.7) Montevideo (3), Senftenberg (2), Emek (1), Hto (1) 10 NAL + NEO + FFN 2 (1.6) Montevideo (2) 11 NAL + NEO + AZM 4 (3.3) Montevideo (1), Senftenberg (1), Emek (2) 12 NAL + NEO + COL 1 (0.8) Montevideo (1) 13 NEO + STR + FOX 3 (2.4) Montevideo (3) 14 NEO + STR + TET 4 (3.3) Montevideo (4) 15 NAL + STR + AZM 2 (1.6) Montevideo (2) 16 NEO + STR + TET + FOX 1 (0.8) spp. (1) 17 NAL + STR + FOX + AZM 1 (0.8) Montevideo (1) 18 NAL + NEO + STR + TET 10 (8.1) Montevideo (10) 19 NAL + NEO + STR + FOX 2 (1.6) Senftenberg (1), spp. (1) 20 NAL + NEO + STR + AZM 1 (0.8) Virchow (1) 21 NAL + NEO + FFN + AZM 1 (0.8) Montevideo (1) 22 NAL + NEO + STR + TET + XNL 2 (1.6) Montevideo (2) 23 NAL + NEO + STR + TET + FOX 2 (1.6) Montevideo (1), Mcclesfield (1) 24 NAL + NEO + STR + TET + AZM 2 (1.6) Montevideo (2) 25 NAL + NEO + STR + FOX + AZM 1 (0.8) Virchow (1) 26 NAL + NEO + STR + FOX + COL + AZM 1 (0.8) spp. (1) 27 NAL + NEO + STR + TET + FOX + AZM 2 (1.6) Montevideo (2) 28 NAL + STR + TET + FFN + AMP + AZM 1 (0.8) Montevideo (1) 29 NAL + NEO + STR + TET + FOX + XNL + AMP 1 (0.8) spp. (1) 30 NAL + NEO + STR + TET + XNL + COL + AZM 1 (0.8) Infntis (1) 31 NAL + NEO + STR + TET + FOX + XNL + AMP + AZM 1 (0.8) Montevideo (1) n, number of isoltes All isoltes showed resistnce to t lest one ntimicrobil, suggesting tht AMR Slmonell is widespred in broiler frms, s ws reported previously in South Kore [33]. Contrry to the incresing prevlence of FQ-resistnt Slmonell reported in other countries [39, 40], we did not observe ny FQ-resistnt isoltes in this study; however, 31.1% of the isoltes showed reduced susceptibility to CIP (with MIC of 0.12 to 0.25 or 0.5 μg/ml), nd 37.4% of the isoltes showed IR to ENR [41, 42]. The rte of IR to CIP ws higher in frms tht used ENR thn in frms tht did not, lthough the difference ws not sttisticlly significnt (p > 0.05). The rtio of IR to ENR ws significntly higher (P < 0.05) in frms tht used ENR thn in those tht did not (Fig. 1c). Resistnce to SXT, TET, nd COL ws significntly higher in frms tht used ENR thn in those tht did not (P < 0.05; Fig. 1c). Resistnce to the three ntimicrobils ENR, TET, nd SXT ws most commonly observed in isoltes from chicken, in previous study [43]. One plusible explntion is tht the selective pressure exerted by the ntimicrobils contributed to the co-selection of this ntimicrobil resistnce pttern. The co-selection of resistnce to more thn one ntimicrobil, owing to the genetic linkge of resistnce genes, is common feture of resistnce cquired by horizontl gene trnsfer [44, 45].
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 8 of 14 Tble 5 Distribution of Slmonell serotypes nd genotypes in broiler frms Frm ENR use Flock Production cycle 1 Production cycle 2 1 dy 15 dys 30 dys 1 dy 15 dys 30 dys A Y 1 S. Montevideo (11) b S. Senftenberg (5) - c S. Montevideo (10) S. Mcclesfield (NC d ) S. Montevideo (8) 2 S. spp. (NC) S. Vellore (NC) B 1 S. Montevideo (7, 8, 15) S. Montevideo (13) S. Montevideo (26) S. Senftenberg (7) 2 S. Montevideo (2, 4) S. Senftenberg (10) S. Montevideo (20) S. Emek (3) S. Mcclesfield (NC) C 1 S. Montevideo (8) S. Montevideo (28) S. Senftenberg (9) S. Emek (4) 2 S. spp. (NC) D 1 S. Senftenberg (2) S. Montevideo (8) S. Montevideo (8, 25) 2 S. Mcclesfield (NC) S. Montevideo (8) S. Montevideo (12) S. Montevideo (8) E 1 S. Montevideo (22) S. Montevideo (5) 2 S. Montevideo (8) S. Montevideo (24) S. Montevideo (3, 13) F 1 S. Virchow (NC) S. Montevideo (30) S. spp. (NC) S. Montevideo (8, 9) 2 S. Montevideo (5) S. Virchow (NC) S. Infntis (NC) G 1 S. Montevideo (17) N e N N 2 N N N H N 1 S. Senftenberg (11) S. Virchow (NC) S. Montevideo (13, 16) S. Senftenberg (4) S. Montevideo (13) S. Mcclesfield (NC) S. Montevideo (13) S. Montevideo (3, 11) 2 S. Senftenberg (8) S. Montevideo (5, 11) S. Montevideo (13) I 1 S. Montevideo (19) S. Montevideo (4) S. Senftenberg (8) S. Emek (2) S. Montevideo (5) S. Edinburg (NC) 2 S. Montevideo (29) S. Montevideo (28) S. Hto (NC) S. Montevideo (28) S. Senftenberg (2) S. Montevideo (16, 28) S. Senftenberg (2) S. Montevideo (28) J 1 S. Montevideo (13) S. Emek (1,2) S. spp. (NC) S. Montevideo (24) S. Montevideo (14, 17, 18) S. Senftenberg (4) S. Montevideo (8) 2 S. Montevideo (11) S. Montevideo (23) S. Montevideo (23) S. Montevideo (13) S. Senftenberg (4) S. Montevideo (14) S. Senftenberg (6)
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 9 of 14 Tble 5 Distribution of Slmonell serotypes nd genotypes in broiler frms (Continued) Frm ENR use Flock Production cycle 1 Production cycle 2 1 dy 15 dys 30 dys 1 dy 15 dys 30 dys K 1 S. Senftenberg (6) S. Montevideo (26) S. Montevideo (22) 2 S. Montevideo (5, 6, 21, 31) S. Montevideo (27) S. Montevideo (22) S. spp. (NC) L 1 S. Senftenberg (NC) S. Senftenberg (NC) N N N 2 N N N ENR use, Y implies yes; N implies no. b PFGE types re showed within prenthesis c The dsh ( ) indictes tht the frm ws negtive for Slmonell d NC implies tht PFGE ws not conducted e N denotes no smpling
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 10 of 14 Fig. 2 Dendrogrms showing pttern nlysis on the bsis of Xb I-PFGE of the 75 Slmonell Montevideo isoltes obtined from broiler frms, long with relted smpling informtion, nd ntimicrobil-resistnce (AMR) profiles. The Dice coefficient ws used to perform similrity nlysis. S, clocl swbs; L, litter; F, feed. b R, resistnce (drk pink); I, intermedite resistnce (pink), S, sensitivity (light pink). Dotted lines indicte 90% similrity In the present study, 17.9% of isoltes showed resistnce to AZM (Tble 3), mcrolide ntimicrobil used for the limited tretment of MDR Slmonell infections [8, 46]. In recent yers, AZM hs been used for typhoid tretment due to the incresed prevlence of MDR Slmonell isoltes, which hs cused serious problems in ntimicrobil selection. The spordic occurrence of AZM-resistnt Slmonell isoltes hs lso cused problems in the selection of suitble ntimicrobils for typhoid tretment [47, 48]. In Europe, the first cse of filure of AZM tretment of Slmonell infection ws reported [49]. Similrly, emergence of clinicl AZM-resistnt Slmonell isoltes ws recently reported in Asi [50, 51]; 15.24% of Slmonell isoltes from chicken showed resistnce to AZM (with n MIC 16 μg/ml) in nother study [52]. However, reports of AZM resistnce in Slmonell isoltes from humns or nimls in South Kore re rre. Considering the fct
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 11 of 14 tht, AZM hs not been used on these frms to the best of our knowledge, the prevlence of AZM resistnce in Slmonell is probbly either due to prolonged use of ntimicrobils in the sme clss s AZM such s erythromycin nd tylosin for treting poultry diseses [53], or due to co-resistnce to AZM nd other ntimicrobils [54]. Further, the MIC 50 of AZM ws 16 μg/ml ginst ll isoltes nd 64 μg/ml ginst S. Virchow (Tble 3). However, the exct reson for the emergence of AZM resistnce in Slmonell nd its mechnism require further studies. Additionlly, becuse COL is lst-line drug used to tret MDR Enterobctericee infections, the four COL-resistnt isoltes identified in the present study wrrnt more ttention; due to the prevlence of COL-resistnt Slmonell, mcr fmily genes cn be esily nd quickly trnsmitted [8, 55, 56]. In Europe, plsmid-medited COL resistnce in Enterobctericee hs lredy spred widely in vin nd pig frms, nd this hs necessitted prompt interntionl ction to restrict or bn COL use in griculture to void further spred of resistnce, similr to the solution involving NDM-1 (New Delhi metllo-β-lctmse-1) severl yers go [56]. COL resistnce in Slmonell isoltes from humns hd not been reported until recently, when the first COL-resistnt isoltes from humns were reported in the Arbin Peninsul [57], followed by two COL-resistnt Slmonell strins in Itly [58]. The observtion of co-resistnce to AZM nd COL in two MDR isoltes (AMR profiles: NAL + NEO + STR + FOX + COL + AZM nd NAL + NEO + STR + TET + XNL + COL + AZM), both of which exhibited intermedite resistnce to CIP nd ENR (dt not shown), is of high importnce. This might pose considerble chllenge when selecting drugs to tret humn Slmonell infections. The most importnt finding of the present study ws tht the AMR profile XNL + COL + AZM, conferring resistnce to the criticlly importnt ntimicrobils used to tret slmonellosis or MDR Slmonell infections in humns, hs never been reported previously in Slmonell strins of niml origin. Bsed on the results of distribution of Slmonell serotypes nd genotypes, considerble cross-contmintion mong the frms could be inferred. For exmple, isoltes with the sme PFGE type shred between flocks, production cycles, smple types, nd mong chickens of different ges within the frms were frequently identified (Fig. 2). This could be becuse the frms shred resources including breeders, trucks for trnsport, veterinrins, chicks, nd feed [59]. Chicks in five frms (frms A, B, D, E, nd J) were from the sme breeder frm which ws negtive for Slmonell (dt not shown). Moreover, there ws contmintion between flocks, production cycles, smple types, nd mong chicken of different ges within the frms. Antimicrobil-resistnt Slmonell cn be circulrly trnsmitted between continuous production cycles. Although ll-in/ll-out replcement systems hve been pplied in the commercil poultry industry, Slmonell contmintion still occurs, especilly in the frm environment. Plusible explntions include insufficient disinfection, development of resistnce to disinfectnts in the first production cycle nd subsequent survivl, nd inherent resistnce in Slmonell [1, 60]. Another explntion is tht other fctors including ir, unclen fcilities, nd vectors such s insects, wild birds, frmers, nd rodents, might contribute to Slmonell trnsmission in poultry frms [61]. The dissemintion of ntimicrobil-resistnt Slmonell in the frms might lso hve occurred vi litter contmintion (frm H; Fig. 2), becuse the litter smples hd higher Slmonell isoltion nd MDR rtes thn the clocl swb nd feed smples (Fig. 1, b), with no significnt difference in isoltion rtes from litter smples between production cycles (Additionl file 2). The spred of indoor Slmonell contmintion in the broiler frms ws pprently cused by litter from the broilers. Slmonell-positive litter smples detected in our study my hve importnt public helth implictions. A recent study reported positive correltion between prevlence of Slmonell in litter smples nd Slmonell isoltion from broiler crcsses [6]. Antimicrobil-resistnt Slmonell could re-circulte in the frms becuse of litter contmintion during different production cycles. Therefore, litter in broiler frms my be n importnt reservoir of Slmonell, consistent with specultion in report from the USA [62]. In summry, we concluded tht serious Slmonell contmintion occurs in frms during production cycles, s does cross-contmintion mong frms owned by the sme compny. Becuse of dissemintion nd cross-contmintion between the frms tht used ntimicrobils nd those tht did not, close ttention should be pid to frm-level hygiene mngement. Conclusion In conclusion, we detected considerble contmintion with ntimicrobil-resistnt Slmonell in broiler frms. The litter in the frm ws one of the importnt reservoirs for Slmonell showing high Slmonell prevlence nd MDR rtes. Other reservoirs of Slmonell, such s feed, ir, fns, nd vectors such s insects, wild birds, frmers, nd rodents might lso contribute to its trnsmission in chicken frms [61]. Additionl mesures for litter nd feed mngement might be required to prevent the trnsmission of ntimicrobil-resistnt Slmonell in such frms. Moreover, ENR use my be n importnt fctor cusing ENR resistnce mong Slmonell in the frms. Our results provide useful informtion regrding the distribution of AMR phenotypes mong
Shng et l. BMC Veterinry Reserch (2018) 14:257 Pge 12 of 14 Slmonell isoltes from broiler frms tht use ENR nd those tht do not, highlighting the need for improved frming prctices nd more cutious use of ntimicrobil gents. Further studies re required to develop protocols to prevent the contmintion of litter nd feed; this, together with instructions for strict ll-in/ll-out replcement nd biosecurity systems, my mrkedly reduce the occurrence of ntimicrobil-resistnt Slmonell species in broiler frms. Additionl files Additionl file 1: Dendrogrms showing pttern nlysis on the bsis of Xb I-PFGE of the 16 Slmonell Senftenberg isoltes obtined from broiler frms nd their ssocition with ntimicrobil-resistnce. The Dice coefficient ws used to perform similrity nlysis. S, clocl swbs; L, litter; F, feed. b R, resistnce (drk pink); I, intermedite resistnce (pink), S, sensitivity (light pink). Dotted lines indicte 90% similrity. (DOCX 271 kb) Additionl file 2: Isoltion rtes nd multidrug resistnt (MDR) rtes in different types of smples obtined during production cycles 1 nd 2. Different lowercse (/b) nd cpitl letters (A/B) in the sme row were used to indicte significnt (P < 0.05) differences in isoltion rtes nd MDR rtes between production cycles 1 nd 2, respectively. (DOCX 36 kb) Abbrevitions AMC: Amoxicillin/clvulnic cid; AMP: Ampicillin; AMR: Antimicrobil resistnce; AZM: Azithromycin; BHI: Brin hert infusion; CHL: Chlormphenicol; CIP: Ciprofloxcin; CLSI: Clinicl Lbortory Stndrdiztion Institute; COL: Colistin; ENR: Enrofloxcin; FDA: Food nd Drug Administrtion; FFN: Florfenicol; FOS: Fosfomycin; FOX: Cefoxitin; FQs: Fluoroquinolones; GEN: Gentmicin; MDR: Multidrug resistnt; MICs: Minimum inhibitory concentrtions; NAL: Nlidixic cid; NDM-1: New Delhi metllo-β-lctmse-1; NEO: Neomycin; PFGE: A pulsed-field gel electrophoresis; RV: Rppport Vssilidis; S. Edinburg: Slmonell Edinburg; S. Emek: Slmonell Emek; S. Hto: Slmonell Hto; S. Infntis: Slmonell Infntis; S. Mcclesfield: Slmonell Mcclesfield; S. Montevideo: Slmonell Montevideo; S. Senftenberg: Slmonell Senftenberg; S. spp.: Slmonell species; S. Vellore: Slmonell Vellore; S. Virchow: Slmonell Virchow; STR: Streptomycin; SXT: Trimethoprim/sulfmethoxzole; TET: Tetrcycline; TIG: Tigecycline; USA: United Sttes of Americ; XLD: Xylose-lysinedeoxycholte; XNL: Ceftiofur Acknowledgements The uthors wish to thnk the veterinry prctitioners nd frmers for their help with smple collection, nd lb members for their help with internl review of the pper. Funding This work ws supported by Kore Institute of Plnning nd Evlution for technology in Food, Agriculture, Forestry nd Fisheries (716002 7, 315035 5), Bsic Science Reserch Progrm through the NRF funded by the Ministry of Eduction (2017R1D1A1B03030883), nd Reserch of Animl nd Plnt Qurntine Agency (Z-1543073-2015-16-01), South Kore. Avilbility of dt nd mterils The dtsets nlyzed in the present study re vilble from the corresponding uthor on resonble request. Additionl supporting files cn be found in the supplementry mteril section. Authors contributions KS nd BW performed experiments, nlyzed the dt nd wrote mnuscript; MK designed the experiments, nlyzed the dt, edited the mnuscript nd coordinted the whole project. All uthors criticlly red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte The current work ws crried out ccording to the ethicl guide lines of Chonbuk Ntionl University (CBNU). Prior to the initition of this study, proper pprovl ws obtined from Ethics Committee for Animl Experiments of the Chonbuk Ntionl University. There re no vulnerble popultions involved, nd no endngered species ws used in the experiments. Frm mngers provided verbl consent to collect the clocl swb smples. Consent for publiction Not pplicble. Competing interests The uthors declre tht they hve no competing interests. 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