Drug Resistance of Enterobacteriaceae from Chicken Carcasses

Transcription

1 1001 Journal of Foo Protection, Vol.60, No.8, 1997, Pages Copyright, International Association of Milk, Foo an Environmental Sanitarians Research Note Drug Resistance of Enterobacteriaceae from Chicken Carcasses Isolate MARIA A.1ESSI,*l MARIA S. SALSI,l MARIA I. CAFFER,2 an MARIA A. MOGUlLEVSKyl l1nstitutoe Tecnologiae Alimentos, Faculta e 1ngenieria Quimica, Universia Nacional el Litoral, C.C 266, 3000 Santa Fe; an 21nstitutoNacional e Microbiologia Dr. C. G. Malbrn, Av. velez Sarsfiel 563, 1281 Buenos Aires, Argentina (MS#96-153: Receive 12 June 1996/Accepte 11 November 1996) ABSTRACT The antibiotic resistance profiles an transferable R factors of Salmonella an Escherichia coli isolates from 104 broiler carcasses taken from one processing plant were etermine. Carcasses were sample after immersion chilling. All samples were transporte ice an immeiately analyze upon arrival to the laboratory. The resistance patterns of isolates to 12 antibiotics were etermine (i.e., ampicillin, cephalothin, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, naliixic aci, tetracycline, neomycin, chloramphenicol, gentamicin, colistin, an nitrofurantoin). Isolates resistant to one or more antibiotics were utilize as onors of resistance to completely antibiotic-sensitive strains, an E. coli K-12, F-, IS, azie-resistant strain an a Salmonella serovar Enteritiis. Transfer of the ifferent R plasmis was confirme by the etermination of the resistance patterns of the transconjugants. Of the 93 Salmonella an 71 E. coli strains isolate from these samples, the largest numbers were resistant to tetracycline (52.7% an 49.3%), sulfisoxazole (45.2% an 42.3%), an streptomycin (37.6% an 39.4%). Large percentages of the Salmonella (33.3%) an the E. coli (30.0%) strains transferre all or part of their resistance to E. coli K-12 in mixe cultures. Great variation was observe between ifferent strains in the frequency at which they transferre resistance. Resistance to tetracycline, sulfisoxazole, an streptomycin was foun to be conferre by 31.7%, 29.8%, an 21.6% of the 19 R factors ientifie. No transfer of resistance to naliixic aci, gentamicin, cephalothin, nitrofurantoin, an chloramphenicol was etecte. When 30 antibiotic-resistant E. coli strains were culture with a sensitive strain of Salmonella serovar Enteritiis,7 (23.3%) ofthe resistant strains were foun capable of transferring R factors. Only 2 (6.7%) of the resistant strains coul transfer R factors an unusual f3-galactosiase activity. Key wors: R factors, Salmonella, E. coli, chicken carcasses The subtherapeutic use of antimicrobial rugs has playe an important role in animal husbanry for control of isease an improvement of growth an efficiency of fee * Author for corresponence. Tel: ; Fax: conversion (9, 14). This agricultural practice introuces selective pressures that potentiate the emergence an istribution of resistant salmonellae in meats an other foo proucts. Poultry appears to be a major reservoir of resistant Salmonella (6). Data have emonstrate that long-term subtherapeutic use in livestock an poultry leas to selection an sprea of transferable multiple resistance between enteric organisms by resistance factors (R factors) (6, 9, 13). Bacterial transmission couple with antibiotic resistance coul have public health implications (4, 9, 14). The present work was performe to stuy the antibiotic resistance profiles an transferable R factors of Salmonella an E. coli isolates from chicken carcasses taken from one processing plant. MATERIALS AND METHODS Chicken carcasses A total of 104 chicken carcasses were obtaine from a commercial processing plant between 1993 an Carcasses were taken from the exit of the chill tank. Four carcasses were sample from each of 26 batches. All samples were transporte in ice containers an analyze immeiately upon arrival to the laboratory. Bacterial strains The bacterial strains use as recipients were the following: E. coli K-12, F-, IS, A;, completely antibiotic sensitive an resistant to 500 flg soium azie per ml, which was obtaine courtesy of Professor L. Le Minor of the WHO Collaborating Centre for Reference an Research on Salmonella, Institut Pasteur, Paris, an Salmonella serovar Enteritiis, sensitive to all antibiotics assaye, which was isolate from poultry. Meia All bacteriological meia were obtaine from Merck Argentina S.A.I.C., except Rappaport-Vassiliais (RV) enrichment meium, prepare in the laboratory following the author's escription (22).

2 1002 TESSI, SALSI, CAFFER, AND MOGUILEVSKY Microbiological analysis of carcasses Salmonella. Twenty-five grams of chicken breast (skin an muscle) was excise, weighe aseptically, an homogenize in 225 ml of buffere peptone water (BPW) in a stomacher an incubate at 35 C for 18 to 24 h (8). Portions (I ml) of pre enrichment cultures were transferre to 10 ml of the following enrichment meia: selenite cystine (SC), Mueller-Kauffmann tetrathionate (TT), an Rappaport- Vassiliais (RV) broths. Selective enrichments were incubate at 35 C (SC an TT) or at 43 C (RV). After 24 h of incubation, a loopful from each selective enrichment broth culture was streake in uplicate on the following selective agar plates: bismuth sulfite (BS), xylose lysine esoxycholate (XLD), an Hektoen enteric (HE). The plates were examine after 24 to 48 hat 35 C. Three typical Salmonella-like colonies were picke from each meium an inoculate into triple sugar iron (TSI) agar an lysine iron agar (LlA). Colonies exhibiting typical reactions on TSI agar an LlA were purifie an further characterize using the urease an oxiase test, followe by API 20 E strips (Analytab Proucts, Inc., Plainview, NY) for all isolates negative for urease an oxiase (1). Biochemically confirme isolates were serologically teste with Salmonella somatic sera. A representative number of Salmonella-positive isolate strains were sent to the National Centre for Reference of Enterobacteria of the Instituto Nacional e Microbiologfa Dr. Carlos O. Malbnin, Buenos Aires, for serological confirmation with somatic an flagellar polyvalent an monovalent antisera. E. coli. Samples were obtaine by premoistening sterile cotton swabs in sterile peptone water (0.1%) (8) an swabbing approximately 100 cm 2 of skin breast region of the carcass. E. coli was isolate on eosin-methylene blue (EMB) agar plates an ientifie accoring to techniques specifie in Bacteriological Analytical Manual (10). Antibiotic resistance profiles Drug resistance of Salmonella an E. coli isolates was etermine accoring to the NCCLS isk iffusion proceure escribe in the National Committee for Clinical Laboratory Stanars (16). The ilute suspension was uniformly inoculate onto the surface of ry Mueller-Hinton (MH) agar plates using an impregnate swab an allowe to ry. Approximately 5 to 6 antimicrobial agent isks were place on each of the inoculate plates using a semiautomate ispenser (Difco Laboratories, Inc., Detroit, MI). The antibiotic test panel consiste of the following agents: ampicillin (AM), 10 Ilg; cephalothin (CF), 30 Ilg; chloramphenicol (C), 30 Ilg; colistin (Col), 10 Ilg; gentamicin (OM), 10 Ilg; naliixic aci (NA), 30 Ilg; neomycin (NE), 30 Ilg; nitrofurantoin (NF), 300 Ilg; streptomycin (S), 10 Ilg; sulfisoxazole (Su), 300 Ilg; tetracycline (TE), 30 Ilg; an trimethoprim an sulfamethoxazole (SxT), 1.25 Ilg an 23.75Ilg. Mueller-Hinton plates were incubate for 16 to 18 h at 35 C. Bacteria were consiere resistant when the clear zone surrouning iniviual antimicrobial isks were the following: $14mm forcf, NF, an TE; $13 mmforam anna; $ 12 mm for C, OM, NE, an Su; $ II mm for Col an S; an $ 10 mm for SxT. Transfer of resistance factors by conjugation The mating proceure escribe in a previous work (18) was use throughout this stuy. Isolates foun to be resistant to one or more antibiotics were utilize as prospective onors of resistance both to a completely antibiotic-sensitive strain, E. coli K-12, F-, J5, A~, an to Salmonella serovar Enteritiis. Resistance to TE was use as a selection marker to select transconjugants from the recipients. To control for recipient mutations, unmate recipient strains were also sprea irectly on selective agar plates containing TE. Both onor an recipient strains were incubate overnight in MH broth at 37 C (12) an ilute to approximately 6 X lobcells per ml by comparison with McFarlan's no. 2 nephelometer stanar (15). One ml of onor broth was mixe with I ml of recipient broth in 4 ml MH broth an incubate for 24 h at 37 C. After incubation the conjugation mixtures were ilute in a series of IO-fol ilutions in 0.85% saline to a 10 7 final ilution. A sample (0.1 ml) from each ilution was sprea-plate onto MacConkey agar plates containing 500 Ilg of soium azie per ml (Sigma Chemical Co., St. Louis, MO; Az-agar plate) to select recipients an one MacConkey agar plate containing 500 Ilg soium azie per ml an 20 Ilg oxytetracycline (TE) per ml (Pfizer S.A.c.I., Argentina; TE-Az-agar plate) to select transconjugants, an the plates were incubate overnight at 37 C. Three typical colonies growing on the selective agar plates were sprea-plate for further isolation an ientifie (1, 10), an their resistance was etermine by the isk metho as previously escribe. RESULTS Incience of rug resistance The numbers of the 93 Salmonella an 71 E. coli isolates from chicken carcasses resistant to the 12 antimicrobial agents are shown in Table 1. Large percentages of Salmonella an E. coli were resistant to tetracycline (52.7% TABLE 1. Antibiotic resistance (%) of Salmonella an E. coli strains isolate from chicken carcasses Number b (%) of resistant bacterial strains Salmonella isolates E. coli Antimicrobial agent" No. (%)C No. (%)C TE 49 (52.7) 35 (49.3) Su 42 (45.2) 30 (42.3) S 35 (37.6) 28 (39.4) NE 10 (10.8) 10 (14.1) AM 20 (21.5) 18 (25.4) SxT 9 (9.7) 9 (12.7) C 8 (8.60) NF 6 (8.5) NA CF Col OM a TE = tetracycline; Su = sulfisoxazole; S = streptomycin; NE = neomycin; AM = ampicillin; SxT = trimethoprim-sulfamethoxazole; C = chloramphenicol; NF = nitrofurantoin; NA naliixic aci; CF = cephalothin; Col = colistin; OM gentamicin. b Total number of isolates teste: Salmonella, 93; E. coli, 71. C Percentage of resistant strains referre to the total number of All isolates were sensitive. _:

3 DRUG RESISTANCE OF ENTEROBACTERIACEAE FROM CHICKEN CARCASSES 1003 TABLE 2. Resistance patterns an transfer characteristics of TABLE 4. Frequency of resistance an R-factor-meiate resisrug-resistant Salmonella strains isolate from chicken carcasses tance to various chemotherapeutic agents in 60 Enterobacteriaceae No. of Resistance R factors strains a isolate from chicken carcasses Resistance resistant pattern Percentage patterna strains transferreb No. (%y of resistant No. of No. of strains strains which TE-Su 3 TE-Su 3 Antimicrobial resistant which transferre transferre TE-Su-S 2 TE-Su-S 2 agent b strains resistance C resistance TE-Su-AM 3 - TE-Su-S-AM 8 TE TE-Su-S-C 6 Su TE-Su-S-AM-NE I TE-S-AM-NE I S TE-Su -S-AM -NE-Sxt 2 TE-Su-S-AM I AM TE-Su-AM-NE 3 TE-Su-AM I NE 18 2 Il.l TE-Su-AM-NE-SxT-C I TE-Su-AM-SxT I SxT TE-Su-S-AM-NE-SxT I TE-Su-S-AM I C 7 Total (33.3) NF 5 a See Table I for explanation of abbreviations. b Recipient antibiotic sensitive strain: E. coli K-12, F-, J 5, A~. _: All isolates were sensitive. a Total numbers of resistant isolates teste: Salmonella, 30; E. coli, 30. b See Table I for explanation of abbreviations. C Recipient antibiotic-sensitive strain: E. coli K-12, F-, J 5, A~. Percentage of R + strains referre to the total number of resistant Enterobacteriaceae for each antimicrobial agent teste. e _: All isolates were sensitive. an 49.3%), sulfisoxazole (45.2% an 42.3%), streptomycin (37.6% an 39.4%), an ampicillin (21.5% an 25.4%). No resistance to gentamicin, naliixic aci, colistin, or cephalothin was etecte. Incience of Rfactors Of the 84 resistant bacteria isolate from chicken carcasses, 30 Salmonella an 30 E. coli strains were ranomly chosen to stuy their ability to transfer their antibiotic resistance to E. coli K-12, F-, 15, A~ (Tables 2 an 3). Our results inicate that antibiotic resistance was eter- mine by transmissible R factors in 10 (33.33%) of the Salmonella an 9 (30.0%) of the E. coli strains. Consierable variation was observe among the ifferent strains in the frequency of resistance transfer. Resistance to tetracycline, sulfisoxazole, an streptomycin was foun to be conferre by 31.7%, 29.8%, an 21.6% of all R factors ientifie (Table 4). When 30 antibiotic-resistant E. coli strains were culture with a sensitive strain of Salmonella serovar Enteritiis, 7 (23.3%) of the resistant strains were foun capable of transferring R factors. Only 2 (6.7%) of the resistant strains TABLE 3. Resistance patterns an transfer characteristics of rug-resistant E. coli strains isolate from chicken carcasses TABLE 5. Resistance patterns an transfer characteristics of rug-resistant E. coli strains isolate from chicken carcasses for transfer to Salmonella serovar Enteritiis No. of Resistance R factors No. of Resistance No. J3-Galac- R Resistance resistant pattern Resistance resistant pattern ofr tosiase factors pattern a strains transferreb No. (%)C pattern a strains transferre b factors activity (%)C TE-Su 5 TE-Su 5 TE-Su 3 TE-Su 3 TE-Su-S-AM 2 TE-S-AM I Te-Su-S 2 TE-Su-Sm I + TE-S-AM-Sxt 3 - TE-Su-AM 2 TE-Su-AM I + TE-Su-NF 2 TE-Su-NE 2 TE-Su I TE-Su-S-AM-NE 8 TE-S-AM-SxT 3 TE-Su-AM-NE 2 TE-Su-AM-NE TE-Su-S-NF 5 TE-Su-S-Sxt I TE-Su-S-SxT TE-Su-S-AM 7 TE-Su-SxT 4 TE-Su-AM-NE 2 TE-Su-AM-NE TE-Su-S-NF 3 TE-Su-S I TE-Su-NE-SxT 4 Total 30 9 (30.0) Total 30 7 (23.3) a See Table I for explanation of abbreviations. b Recipient antibiotic sensitive strain: E. coli K-12, F-, J 5, A~. _: All isolates were sensitive. a See Table I for explanation of abbreviations. b Recipient antibiotic-sensitive strain: Salmonella serovar Enteritiis. All isolates were sensitive. _:

4 1004 TESSI, SALSI, CAFFER, AND MOGUILEVSKY were foun capable of transferring both R factors an unusual ~-galactosiase activity (Table 5). DISCUSSION Our results inicate that R factors play an important role in the resistance of the Salmonella an E. coli strains we isolate from chicken carcasses sample immeiately after chilling. Antibiotic-resistant E. coli strains isolate from these samples pose a concern not only with respect to transfer ofr factors but also regaring the possible transmission of ~-galactosiase activity to Salmonella serovar Enteritiis (Table 5). Transmission of such activity can have a confouning influence on the conventional biochemical methos for the ientification of Salmonella. The prevalence of chloramphenicol-resistant (8.60%) an ampicillin-resistant (21.5%) Salmonella strains in chicken carcasses (Table 1) is isquieting because these antibacterial agents together with SxT (for which resistance was foun in 9.70% of the Salmonella strains) are wiely use in the treatment of human systemic salmonellosis (6). Data have emonstrate that human infections with rug-resistant bacteria carry twice the risk of severe isease, hospitalization, an eath compare to rug-susceptible strains, unerscoring the importance of the issue of antibiotic-resistant Salmonella strains (11). The resistance of Salmonella an E. coli strains to TE, Su, an S was also revealing (Table 1). The intense husbanry practices use in the poultry inustry an the wiesprea use of meicate fees in broiler an layer operations facilitate the horizontal transmission of salmonellae within flocks an encourage the sheing of resistant strains in the poultry environment (6). Contamination of chicken carcasses uring slaughtering proceures is unesirable but unavoiable. The skinning an evisceration steps are major opportunities for contamination (7, 21). Chill water may be an important route of crosscontamination of carcasses with Enterobacteriaceae (or other possible enteric bacteria) with multiple antibiotic resistances (2, 17, 19, 21). A variety of methos have been evelope to reuce the levels of contaminating bacteria on chicken carcasses (7, 19,21). Antibiotic-resistant bacteria represent a persistant pool of resistance genes that are potentially transferable to human pathogens (13). Ientification an classification of plasmis present in these bacteria can be use to trace the source of an infection, an plasmis can serve as strain markers in epiemiological investigations (3, 5). There is general agreement that the pool of resistance genes in the environment can be amplifie by the use of antimicrobial agents. 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