Print version ISSN 1516-8913
Braz. arch. biol. technol. vol.47 no.2 Curitiba June 2004
HUMAN AND ANIMAL HEALTH
Elaine Regina DelicatoI; Jane Martha Graton MikchaII; Sueli Aparecida FernandesIII; Jacinta Sanchez PelayoI, *
IDepartamento de Microbiologia; CCB; Universidade Estadual de Londrina; 86051-990; Londrina - PR - Brazil
IIDepartamento de Análises Clínicas; Universidade Estadual de Maringá; 87020-900; Maringá - PR - Brazil
IIISeção de Bacteriologia; Instituto Adolfo Lutz; 01246-902;São Paulo - SP - Brazil
The purpose of this study was to analyse the profile of antimicrobial resistance among 21 strains of Salmonella isolated from patients with gastroenteritis symptom. It was observed that S. enteritidis was the serotype prevalent. These strains were sensitive to the majority of the antimicrobials tested, however, high resistance was observed in S. typhimurium and S. enterica subsp. enterica serotype 4,5,12:i:-. Surveillance and an efficient monitoring should be priority for the public health for the containment of antimicrobial resistance in foodborne infections.
Key words: Salmonella, humans, serotypes, resistance, antimicrobials
O objetivo deste estudo foi analisar o perfil de resistência à antimicrobianos, de Salmonella isoladas de 21 amostras provenientes de pacientes com sintomatologia de gastroenterite. Verificou-se que S. enteritidis foi o sorotipo com maior prevalência. Estas cepas mostraram-se sensíveis à maioria dos antimicrobianos testados, no entanto, as cepas S. typhimurium e S. enterica subsp. enterica sorotipo 4,5,12:i:-, mostraram-se resistentes à vários antimicrobianos. Vigilância e um monitoramento eficiente, para diminuir a resistência antimicrobiana em microrganismos que causam infecções veiculadas por alimentos, devem ser prioridade para a saúde pública.
Salmonella bacterium is one of the commonest causes of food poisoning worldwide. Food, such as egg, meat, milk and other dairy products, are the commonest source of salmonella for humans (Winokur et al., 2000). The Salmonella genus belongs to the Enterobacteriacea family, according to current taxonomy, and consists of two species: S. enterica and S. bongori. Salmonella enterica is divided into six subspecies: enterica, salamae, arizonae, indica, diarizonae and houtenae. These species and subspecies can be classified according to the identification of antigenic factors (O-somatic, Vi-capsular and H- flagellar) in about 2400 serotypes (Popoff and Le Minor 1992). S. enteritidis is the most common serotype in Brazil and in the world. An increase in the number of cases of diseases in humans caused by food contaminated by this serotype has been observed, but most of the isolates have been sensitive to the antimicrobials chosen for treatment of systemic salmonellosis (Bäumler et al., 2000; Tavechio et al., 1999; Fernandes et al., 1999; Reis et al., 1999). Although most Salmonella isolates belong to the Enteritidis serotype, S. typhimurium has increased resistance dissemination to antimicrobials, especially S. typhimurium phage type 104 (DT104) that has chromosome resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide and tetracycline (ACSSuT) (Gross et al., 1998; Glynn et al., 1998). The aim of this study was to verify the resistance profile to antimicrobials among Salmonella serotypes isolated from patients in two major hospitals in Londrina-PR.
MATERIALS AND METHODS
Twenty-one strains of Salmonella spp. were isolated in two major hospitals in the city of Londrina-PR, between October 1999 and May 2000. They were isolated from feces, except one that was isolated from blood. Feces were collected in clean recipients and blood in hemoculture flask.
Isolation and biochemical identification
The fecal samples were inoculated in Hektoen-Enteric agar (Merck), MacConkey agar (Merck), and enriched Selenite-Cystine broth (Merck) followed by incubation for 18-24 hours at 37oC. The colonies that grew on both solid media were inoculated in culture medium for biochemical identification: EPM, MILi (Toledo et al., 1982 a,b) and Simmons Citrate (Merck), and also incubated for 18-24 hours at 37oC. Selenite-Cystine broth was inoculated in MacConkey agar and Salmonella-Shigella agar (Merck) and incubated for 18-24 hours at 37oC. Biochemical identification was then done, as previously mentioned. The blood sample was inoculated onto Blood agar and MacConkey agar, and the colonies isolated from MacConkey agar were biochemically identified as described above.
Strains with Salmonella biochemical characteristics were confirmed with polyvalent anti-Salmonella serum (Probac do Brasil, São Paulo, Brazil) and those positive were sent to the Instituto Adolfo Lutz, São Paulo, for serotyping.
The antimicrobial susceptibility was determined by the disk diffusion method on Müller-Hinton agar (Biobrás), as described by Bauer et al. (1966). Initially, 16 antimicrobials were tested; those which corresponded to the most routinely used in clinical laboratory, such as, amikacin, ampicillin, aztreonam, carbenicillin, cephalotin, cefepime, cefotaxime, cefoxitin, ceftriaxone, ciprofloxacin, chloramphenicol, gentamicin, imipenen, pefloxacin, trimethoprim+sulfamethoxazole and tobramycin. The interpretation of the inhibition halos to the antimicrobials was according to the National Committee for Clinical Laboratory Standards (NCCLS, 2000a). When the results were analyzed it was suspected that four strains might contain the phage type 104 (ACSSuT phenotype) because they presented resistance to two antimicrobials reported to this phenotype (ampicillin and chloramphenicol). Therefore, the resistance profiles of these strains were evaluated for streptomycin, sulfonamide and tetracycline in addition.
The results showed that S. enteritidis was the most frequently isolated serotype (66.7%). The other serotypes, S. infantis, S. newport, S. typhimurium, S. javiana, S. brandenburg , S. glostrup and S. enterica subsp. enterica serotype 4,5,12:i:- were isolated at frequency of 4.76%. All the serotypes were isolated from feces except S. enterica subsp. enterica serotype 4,5,12:i:- that was isolated from blood. Table 1 shows the resistance profiles to 16 antimicrobials initially tested. Seven Salmonella strains (50%) belonging to Enteritidis serotype were sensitive to all the antimicrobials tested and the other seven were resistant to one or more antimicrobials, and the highest percentage of resistance was found for ampicillin (85.7%). S. infantis, S. newport, S. javiana and S. brandenburg were sensitive to all the antimicrobials, and S. glostrup was resistant only to gentamicin. However, two strains belonging to Typhimurium serotype and enterica serotype 4,5,12:i:- were resistant to 11 antimicrobials. All strains tested were sensitive to amikacin, pefloxacin, imipenen and ciprofloxacin.
Four strains (two S. enteritidis, one S. typhimurium, and one S. enterica subsp. enterica serotype 4,5,12:i:-) that were suspected of containing the phage type 104 inserted in their chromosome, presented resistance to ampicillin, chloramphenicol, streptomycin and sulfonamide but were sensitive to tetracycline.
The data obtained for the frequency of Salmonella strains isolated were according to the literature, and showed prevalence of S. enteritidis (Bäumler et al., 2000, Tavechio et al., 1999; Fernandes et al., 1999; Reis et al., 1999). Bäumler et al. (2000) attributed the increase of salmonellosis cases by this serotype because of the replacement of S. pillorum and S. gallinarum in an ecological niche that was previously occupied by the last two serotypes of avian origin. These three serotypes have a common antigen (O9) and the immune response produced by the avian Salmonella strains did not allow birds to be infected by the Enteritidis serotype. By eradication of these serotypes, at the beginning of the second half of the last century, S. enteritidis started to infect birds and consequently humans.
According to the resistance patterns to the antimicrobials tested, it was found that 50% of the S. enteritidis isolates were sensitive to all the antimicrobials and that the other 50% presented resistance to one or more antimicrobials. Attention was drawn to strains resistant to ampicillin, trimethoprim+sulfamethoxazole, chloramphenicol and third generation cefalosporine because the NCCLS (2000b) recommended that only ampicillin, a quinolone and trimethoprim+sulfamethoxazole be tested and reported for the Salmonella isolates of intestinal infection and a third generation cefalosporine and chloramphenicol should be tested and reported only for extraintestinal infections. According to our results, three strains of S. Enteritidis were resistant to ampicillin, one to ampicillin and chloramphenicol, one to ampicillin and trimethoprim+sulfamethoxazole and other to ampicillin, trimethoprim+sulfamethoxazole and chloramphenicol. S. typhimurium and S. enterica subsp. enterica serotype 4,5,12:i:- were resistant to third generation cefalosporine, ampicillin and chloramphenicol. Therefore, monitoring programs are needed to detect these resistant strains before they become widely disseminated. The current concern has been the isolation of S. enteritidis strains that produce Extended Spectrum Beta Lactamase (ESBL) capable of hydrolyzing first, third and fourth generation cephalosporine and monobactamic, but do not act on cefamicines (second generation cefalosporine) (Nastasi et al., 2000; Rankin and Coyne, 1998; Ling et al., 1998). No S. enteritidis strain with this resistance mechanism was isolated in this study. A second resistance mechanism has recently been detected mainly in Typhimurium serotype, concerned to a Beta Lactamase type Amp-C mediated by plasmid, conferring additional resistance to cefamicines (Winokur et al. 2000).
In the present study, our strains of S. typhimurium and S. enterica subspecies enterica serotype 4,5,12:i:- presented a multiresistance pattern and among these resistance patterns were detected from first to fourth generation cefalophorines, such as cephalotin (1st generation); cefoxitin (2nd generation); cefotaxime and ceftriaxone (3rd generation); cefepime (4th generation), and also to the monobactamic aztreonam. Therefore, it could be suggested that phenotypic and genotypic tests should be done to detect ESBL and Amp-C in these strains. It has been mentioned in the literature the concern about Salmonella strains that may contain phage type 104 (ACSSuT phenotype). In this study of the four strains that were suspected of containing this phage, all presented the ACSSu phenotype, being sensitive to tetracycline. A contributing factor for resistance dissemination observed in certain Salmonella serotypes has been the unnecessary use of antimicrobials for self limited gastroenteritis treatment. It has been suggested that these treatments be reserved only for extraintestinal salmonellosis and in cases of child gastroenteritis or elderly and immune suppressed patients where antibioticotherapy is fundamental to control these diseases (Winokur et al., 2000; Prats et al., 2000). Regarding antimicrobials resistance of enteric pathogens, the most worrying factor has been the increase of multiresistance dissemination of S. typhimurium and also, on a smaller scale, by other serotypes due to the use of these antimicrobials in animal feeding for prophylaxis and growth promotion. Therefore, it is expected that prophylactic measures will soon be introduced to decrease the use of antimicrobials in products of animal origin, which certainly will result in a decline in strains resistance to antimicrobials in the food chain (Threlfall et al., 2000).
We thank Prof. Carlos Nozawa for revising the manuscript.
Bauer, A. W.; Kirby, W. M. M.; Sherris, J. C. and Tuck, M. (1966), Antibiotic susceptibility testing by standardized single disk methods. Am. J. Clin. Pathol., 45, 494-496. [ Links ]
Baümler, A. J.; Hargis, B. M. and Tsolis, R. M. (2000), Trancing the origins of Salmonella outbreaks. Science,287, 50-52. [ Links ]
Fernandes, S. A.; Tavechio, A. T.; Yonamine, E. K.; Ghilardi, A. C. R.; Valle, G. R. F.; Araujo E. A. C.; Almeida, I. A. Z. C. and Irino, K. (1999), Sorotipos de Salmonella prevalentes em infecções humanas, no estado de São Paulo: 1994-1998. Paper presented in 20. Congresso Brasileiro de Microbiologia, 24-28 October, Salvador, Bahia. [ Links ]
Gross, U.; Tschape, H.; Bednarek, I. and Frosch, M. (1998), Antibiotic resistance in Salmonella enterica sorotipo typhimurium. Eur. J. Clin. Microbiol. Infect. Dis., 17, 385-387. [ Links ]
Glynn, M. K.; Bopp, C.; Dewitt, W.; Dabney, P.; Mokhtar, M. and Angulo, F. J. (1998), Emergence of multidrug-resistant Salmonella enterica serotype typhimurium DT104 infections in United States. N. Engl. J. Med., 338, 1333-1338. [ Links ]
Ling, J. M.; Koo, I. C.; Kam, K. M. and Cheng, A. F. (1998), Antimicrobial susceptibilities and molecular epidemiology of Salmonella enterica sorotipo enteritidis strains isolated in Hong- Kong from 1986 to 1996. J. Clin. Microbiol., 36, 1693-1699. [ Links ]
Nastasi, A.; Mammina, C. and Cannova, L. (2000), Antimicrobial resistance in Salmonella Enteritidis, Southern Italy, 1990-1998. Emerg. Infect. Dis.,6, 401-403. [ Links ]
National Committee for Clinical Laboratory Standards. (2000a), Performance standards for antimicrobial disk susceptibility tests, aproved standard. 7th ed. NCCLS Document M2-A7. Wayne, Pa. [ Links ]
National Committee for Clinical Laboratory Standards (2000b), Mic testing. NCCLS Document M100-S10. Wayne, Pa. [ Links ]
Prats, G.; Mirelis, B.; Llovet, T.; Munoz, C.; Miró, E. and Navarro, F. (2000), Antibiotic resistance trends in enteropathogenic bacteria isolated in 1985-1987 and 1995-1998 in Barcelona. Antimicrob. Ag. Chemother., 44, 1140-1145. [ Links ]
Popoff, M. Y. and Le Minor, L. (1992), Antigenic formulas of the Salmonella serovars. 6 th ed. Paris, France: Who Collaborating Centre for Reference and Research on Salmonella, Institut Pasteur. [ Links ]
Rankin, S. C and Couyne, M. J. (1998), Multiple antibiotic resistance in Salmonella enterica serotype enteritidis. Lancet,351, 1740. [ Links ]
Reis, E. M. F.; Gomes, E. D. T.; Filho, S. P.; Feitosa, D. P.; Costa, R. G.; Rodrigues, D. P. and Hofer, E. (1999), Sorovares prevalentes de Salmonella isolados de diferentes regiões do país e fontes de infecção no período de janeiro 1998 a junho 1999. Paper presented in 20. Congresso Brasileiro de Microbiologia, 24-28 October, Salvador, Bahia. [ Links ]
Tavechio, A. T.; Yonamine, E. K.; Ghilardi, A. C. R.; Valle, G. R. F.; Arruda, A. C.; Irino, K. and Fernandes, S. A. (1999), Resistência antimicrobiana de sorotipos de Salmonella isolados no estado de São Paulo, no período de 1996 a 1999. Paper presented in 20. Congresso Brasileiro de Microbiologia, 24-28 October, Salvador, Bahia. [ Links ]
Threlfall, E. J.; Ward, L. R.; Frost J. A.; and Willshaw, G. A. (2000), The emergence and spread of antibiotic resistance in food-borne bacteria. Int. J. Food Microbiol., 62, 1-5. [ Links ]
Toledo, M. R. F.; Fontes, C. F., and Trabulsi, L. R. (1982a), MILi: um meio para a realização dos testes de motilidade, indol e lisina descarboxilase. Rev. Microbiol.,13, 230-235. [ Links ]
Toledo, M. R. F.; Fontes, C. F., and Trabulsi, L. R. (1982b), EPM: uma modificação do meio de Rugai para a realização simultânea dos testes de produção de gás a partir de glicose, H2S, urease, e triptofano desaminase. Rev. Microbiol., 13, 309-315. [ Links ]
Winokur, P. L.; Brueggemann, A.; DeSalvo, D. L.; Hoffmann, L.; Apley, M. D., Uhlenhopp, E. K.; Peafller, M. A. and Doern, G. V. (2000), Animal and human multidrug-resistant, cephalosporin- resistant Salmonella isolates expressing a plasmid-mediated CMY-2 AmpC ß-lactamase. Antimicrob. Ag. Chemother.,44, 2777-2783. [ Links ]
Received: July 03, 2002
Revised: June 04, 2003
Accepted: August 15, 2003
* Author for correspondence