MICROBIAL DRUG RESISTANCE Volume 8, Number 4, 2002 © Mary Ann Liebert, Inc. Antimicrobial Resistance of Salmonella Serotypes Isolated from Slaughter-Age Pigs and Environmental Samples CELSO JOSÉ BRUNO OLIVEIRA,1 LUIZ FERNANDO OLIVEIRA SILVA CARVALHO,2 SUELI APARECIDA FERNANDES,3 ANA TEREZINHA TAVECHIO,3 CLAUDIO CAMACHO PEREIRA MENEZES,2 and FRANCISCO JOSÉ DOMINGUES, JR.2 ABSTRACT The aim of this study was to determine the antimicrobial resistance patterns of Salmonella strains isolated from slaughter-age pigs and environmental samples collected at modern swine raising facilities in Brazil. Seventeen isolates of six serotypes of Salmonella enterica subsp. enterica were isolated out of 1,026 collected samples: Salmonella Typhimurium (1), Salmonella Agona (5), Salmonella Sandiego (5), Salmonella Rissen (1), Salmonella Senftenberg (4), and Salmonella Javiana (1). Resistance patterns were determined to extended- spectrum penicillin (ampicillin), broad-spectrum cephalosporins (cefotaxime and ceftriaxone), aminoglyco- sides (streptomycin, neomycin, gentamicin, amikacin, and tobramycin), narrow-spectrum quinolone (nalidixic acid), broad-spectrum quinolone (ciprofloxacin and norfloxacin), tetracycline, trimethoprim, and chloram- phenicol. Antimicrobial resistance patterns varied among serotypes, but isolates from a single serotype con- sistently showed the same resistance profile. All isolates were resistant to tetracycline, streptomycin, and nalidixic acid. One isolate, Salmonella Rissen, was also resistant to cefotaxime and tobramycin. All serotypes were susceptible to ceftriaxone, norfloxacin, ciprofloxacin, ampicillin, gentamicin, and chloramphenicol. The high resistance to tetracycline and streptomycin may be linked to their common use as therapeutic drugs on the tested farms. No relation was seen between nalidixic acid and fluoroquinolone resistance. 407 INTRODUCTION NONTYPHOID AL SALMONELLOSIS has become an important cause of enteric infections in many countries,17,23,30 and several human salmonellosis outbreaks linked to the consump- tion of contaminated pork have been reported during the last decade.3,20,22,24,29 Furthermore, antibiotic-resistant strains have emerged worldwide and have led to increasing consumer con- cern about animal-derived food consumption. Infections caused by resistant strains have shown a higher fatality rate compared to those caused by susceptible organisms.15 The extensive use of antibiotics on farms for both therapeu- tic and growth promotion purposes have been considered to be an important trigger for the emergence of antibiotic-resistant pathogens,9,14,33 and their real impact on public health contin- ues to be a matter of debate. High numbers of tetracycline-re- sistant Salmonella strains have been isolated from pig farms.7,34 However, resistance to first-choice antibiotics used for treat- ment of human infections causes even more concern. An in- creasing number of reports on Salmonella isolates from animals and humans show reduced susceptibility to fluoro- quinolones.2,31,35 Also, resistance to third-generation cephalo- sporins has been recently published.4,5 Considering the marked importance of Salmonella organ- isms as food-borne pathogens and the world-wide emergence of resistant or multidrug-resistant Salmonella strains,6,33 in the present paper we describe the antimicrobial resistance patterns of Salmonella serotypes isolated from pigs and environmen- tal samples collected from modern swine-raising systems in Brazil. 1Departamento de Patologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Unesp campus de Jaboticabal, 14 884-900, Jaboticabal, SP, Brasil. 2Departamento de Clínica e Cirurgia Veterinária da Faculdade de Ciências Agrárias e Veterinárias, Unesp campus de Jaboticabal, Jabotica- bal, SP, Brazil 14 884-900. 3Instituto Adolfo Lutz, setor de Bacteriologia, Bairro Cerqueira César, São Paulo-SP, Brazil 01246-902. MATERIALS AND METHODS Antibiotic resistance patterns were studied in 17 Salmonella strains previously isolated from four pig farms located in São Paulo State, Brazil (Oliveira et al., manuscript submitted). Six- teen positive samples were isolated out of 1,026 collected sam- ples and the serotypes of Salmonella enterica subsp. enterica comprised Salmonella Typhimurium (1), Salmonella Agona (5), Salmonella Sandiego (5), Salmonella Rissen (1), Salmo- nella Senftenberg (4), and Salmonella Javiana (1). Sixteen pos- itive samples were identified out of all samples collected, in- cluding feces, feed, floors, feeders, and water. Two different serotypes (S. Agona and S. Sandiego) were cultured from a sin- gle positive fecal sample. Bacteriological procedures for feces and water samples were followed as previously described.1,12 Floor and feeder samplings were performed after the animals were removed and the pens disinfected, using drag swab sets.21 The isolated Salmonella strains were serotyped according to Popoff and Le Minor.27 Resistance to each antibiotic was determined by the Kirby-Bauer disk susceptibility test28 using commercially prepared filter paper disks (Oxoid Ltd., Hamp- shire, UK), each impregnated with a specified amount of an antibiotic. The antibiotics tested and respective breakpoints were as follows: extended spectrum penicillin (ampicillin, $32 mg/ml); broad-spectrum cephalosporins (cefotaxime, $64 mg/ml; ceftriaxone, $64 mg/ml); aminoglycosides (strep- tomycin, $2,000 mg/ml; neomycin, $16 mg/ml; gentamicin, $16 mg/ml; amikacin, $64 mg/ml; tobramycin, $16 mg/ml); narrow-spectrum quinolone (nalidixic acid, $32 mg/ml); broad- spectrum quinolones (ciprofloxacin, $4 mg/ml; norfloxa- cin, $16 mg/ml; tetracycline, $16 mg/ml; trimethoprim, $16 mg/ml; chloramphenicol, $32 mg/ml). The results were analyzed and interpreted according to the guidelines of the Na- tional Committee for Clinical Laboratory Standards (NC- CLS).25 Briefly, after pre-inoculation in tryptone soy broth and adjustment of turbidity to a 0.5 McFarland turbidity standard, bacterial suspensions were streaked onto freshly prepared Mueller-Hinton agar plates (Oxoid Ltd., Hampshire, UK) using sterile swabs. The reference strains used were Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Seven disks were placed on each 150-mm plate after 5 min to allow any excess surface moisture to be absorbed, and plates were aerobically incubated for 16–18 hr at 35°C. The strains were finally interpreted as resistant, intermediate or sus- ceptible to each antibiotic according to the criteria currently rec- ommended by the NCCLS, based on the diameters of the zones of complete inhibition to the nearest whole millimeter. RESULTS Seventeen Salmonella strains were isolated out of 1,026 sam- ples collected. The Salmonella serotypes identified and the types of samples from which they were detected are shown in Table 1. The antimicrobial resistance patterns are shown in Table 2. Susceptibility varied among serotypes, but isolates from a single serotype showed the same resistance pattern. All isolates were resistant to at least three antimicrobial agents, tetracycline, streptomycin, and nalidixic acid. One isolate, Sal- monella Rissen, was also resistant to cefotaxime and to- bramycin. All serotypes were susceptible to ceftriaxone, nor- floxacin, ciprofloxacin, ampicillin, gentamicin, and chloramphenicol. Fifteen isolates showed intermediate resis- tance to neomycin. Intermediate resistance to amikacin (3), trimethoprim (3), nalidixic acid (1), tobramycin (1), and cefo- taxime (1) was detected as well. The serotypes from farm A were isolated from different types of samples; however, a very similar antimicrobial resistance profile was seen for these strains (Table 2). DISCUSSION Considering the marked importance of Salmonella enterica serotypes as food-borne pathogens19 and the world-wide emer- gence of resistant or multidrug-resistant Salmonella strains,16,33 our goal was to report the antibiotic resistance profiles of Sal- monella strains isolated from pigs and environmental samples collected from modern swine raising systems in Brazil. Despite the marked limitation of this study due to the small number of isolates (16 out of 1,026 samples), some results may be dis- cussed. All Salmonella isolates collected from four pig farms showed resistance to tetracycline and streptomycin. Although these an- tibiotics are not allowed for use as growth promoters by Brazil- ian legislation, they have been used by veterinarians for clini- OLIVEIRA ET AL.408 TABLE 1. NUMBER OF SALMONELLA SEROTYPES CULTURED FROM SWINE FARMS A, B, C, AND D, LOCATED IN SÃO PAULO STATE (BRAZIL), CONSIDERING THE TYPES OF SAMPLES FROM WHICH THEY WERE ISOLATED Test farm Serotype (number of isolates) Samples (number of positive samples) Salmonella Agona (3) Rectal feces (1), dunging gutter water (2) Salmonella Agona (1) Rectal feces (1) A Salmonella Sandiego (4) Pool of feces (2), feed (1) drinking water (1) Salmonella Javiana (1) Rectal feces (1) Salmonella Agona (1) 1 Salmonella Sandiego (1)a Rectal feces (1) B Salmonella Rissen (1) Rectal feces C Salmonella Senftenberg (2) Rectal feces (2) Salmonella Senftenberg (2) Rectal feces (2), pool of feces (2) D Salmonella Typhimurium (1) Clean feeder (1) aIsolation of two serotypes from one fecal sample. cal therapy on the tested farms. Furthermore, high frequencies of isolates resistant to tetracycline have been also detected in Salmonella isolates of swine origin collected in other coun- tries.7,34 In the United States, 84.2% of Salmonella strains iso- lated from swine were found to be resistant to tetracycline.7 It is likely that a link might exist between the long-term thera- peutic use of certain antibiotics and the high number of isolates showing resistance patterns to them. Furthermore, the use of antimicrobials has been implicated in the emergence and main- tenance of resistant determinants in various ecosystems.9,22,26 However, the real impact of the therapeutic use of these an- tibiotics in farm animals on the spread of resistance remains to be clarified. We detected Salmonella Rissen resistant to cefotaxime. Some recent reports4,5,8 also showed resistance of other Sal- monella strains to third-generation cephalosporins. Despite the importance of expanded-spectrum cephalosporins in the treat- ment of invasive salmonellosis in children, the incidence of Sal- monella resistant to them remains very low. No resistance to cefotaxime was detected in 1,257 Salmonella isolates from the United States.7 Furthermore, Salmonella Rissen has been rarely isolated from human sources in Brazil. In a retrospective study on Salmonella isolates from human sources in Brazil from 1991 to 1995,17 only 4 Salmonella Rissen strains were isolated from human sources compared to 200 strains of Salmonella Ty- phimurium. For these reasons, we believe that the isolation of a cefotaxime-resistant Salmonella Rissen strain may not be a direct concern to human health but could be an important ob- servation because such resistance could be transferred to other important pathogenic serotypes. Treatments failed when fluoroquinolones were used to treat Salmonella infections caused by nalidixic acid-resistant strains.13,24 Indeed, Salmonella isolates resistant to nalidixic acid have been shown to be less susceptible to fluoro- quinolones.35,36 Strains showing decreased susceptibility to ciprofloxacin, ofloxacin, and norfloxacin have been reported to be uniformly resistant to nalidixic acid, a fact attributed to a mutation in the gyrA gene.10,11 Conversely, our results showed that all strains were nalidixic acid resistant but norfloxacin and ciprofloxacin susceptible, corroborating data previously re- ported in The Netherlands.18 Therefore, no link between nalidixic acid and fluoroquinolone resistance was detected. We believe this is an important point because fluoroquinolone re- sistance has received special attention from the scientific com- munity. Many reports have indicated a possible link between the increased recovery of Salmonella organisms showing re- duced susceptibility to ciprofloxacin and the importance of us- ing quinolones in farm animals, with the consequent emergence of fluoroquinolones resistance.10,32 Curiously, all Salmonella serotypes from farm A showed a very similar resistance profile, despite the fact that the serotypes isolated may develop antibiotic resistance through different mechanisms.35 This result may indicate that horizontal gene transfer between bacteria is important to resistant genes spread- ing in a given environment, supporting previous reports.15 How- ever, conclusions should be reached with caution because all serotypes were theoretically submitted to the same antimicro- bial selective pressure on such farm. Further work looking at the genetic characterization of resistance determinants is un- derway to determine the identity and homology of resistance genes. The isolation of a multiresistant Salmonella Typhimurium strain from a clean and supposedly disinfected feeder may in- dicate failures of biosecurity procedures that play an important role in the introduction and maintenance of resistant serotypes on farms. Nevertheless, further studies of the risk factors that might contribute to the maintenance of resistant Salmonella strains are needed. There is a one-way trend worldwide to implement changes in pig production toward health-oriented systems. This is a cru- cial attitude for the reduction of antibiotic use on farms, since the benefits of such drugs are emphasized in systems with low hygienic standards.9 Several antibiotics earlier used as growth promoters have been banned in Brazil and at this time only 14 antibiotics are allowed for use on pig farms for this purpose. Furthermore, special attention has been paid to the therapeutic use of antibiotics for farm animals in this country. Finally, we also assume that the implementation and im- ANTIMICROBIAL RESISTANCE OF SALMONELLA FROM PIGS 409 TABLE 2. ANTIMICROBIAL RESISTANCE PATTERNS FROM 17 SALMONELLA STRAINS ISOLATED FROM DIFFERENT TYPES OF SAMPLES COLLECTED ON INTENSIVE PIG FARMS LOCATED IN SÃO PAULO , BRAZIL Serotype Intermediate Sample (number of isolates) Total resistance pattern resistance pattern Rectal feces Salmonella Agona (3) Str/Tet/Nal Tm/Neo Salmonella Rissen (1) Ctx/Str/Tet/Tob Tm/Nal/Neo Salmonella Senftenberg (2) Str/Tet/Nal Ami/Neo/Tob Salmonella Javiana (1) Str/Tet/Nal Neo Salmonella Sandiego (1) Str/Tet/Nal Neo Feed Salmonella Sandiego (1) Str/Tet/Nal Neo Pool of feces Salmonella Senftenberg (2) Str/Tet/Nal Ami/Neo/Tob Salmonella Sandiego (2) Str/Tet/Nal Neo Drinking water Salmonella Sandiego (1) Str/Tet/Nal Neo Clean feeder Salmonella Typhimurium (1) Str/Tet/Nal Ctx/Ami Dunging gutter water Salmonella Agona (2) Str/Tet/Nal Tm/Neo The antimicrobial agents tested were: cefotaxime (Ctx), ceftriaxone (Cro), amikacin (Ami), norfloxacin (Nor), streptomycin (Str), trimethoprim (Tm), neomycin (Neo), tobramycin (Tob), ciprofloxacin (Cip), tetracycline (Tet), ampicillin (Amp), genta- micin (Gen), nalidixic acid (Nal), and chloramphenicol (Chl). provement of biosecurity procedures on pig farms have a great potential to prevent the maintenance of resistant strains through internal cycles of contamination. 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Carvalho Departamento de Clínica e Cirurgia Veterinária da Faculdade de Ciências Agrárias e Veterinárias-Unesp Campus de Jaboticabal Via de acesso Prof. Paulo Donato Castellane s/n Jaboticabal, SP, Brazil 14 884-900 E-mail: LFOSC@fcav.unesp.br ANTIMICROBIAL RESISTANCE OF SALMONELLA FROM PIGS 411