Arq01_153-04

Brazilian Journal of Microbiology (2007) 38:599-602ISSN 1517-8382 ISOLATION OF SHIGA TOXIGENIC ESCHERICHIA COLI FROM BUTCHERIES IN
TAQUARITINGA CITY, STATE OF SÃO PAULO, BRAZIL
Daniela Rodolpho1; José Moacir Marin2*
1Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Universidade Estadual Paulista, SP, Brasil; 2Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil Submitted: January 3, 2007; Returned to authors for corrections: May 31, 2007; Approved: September 28, 2007.
ABSTRACT
Shiga toxigenic Escherichia coli (STEC) has been implicated as the cause of several human diseases. Samples(ground beef, grinding-machines and human hands) from 23 butcheries were assayed for E. coli usingstandard microbiological methods, and 287 isolates were submitted to polymerase chain reaction for thedetection of stx 1, stx 2 and eae genes. Four STEC isolates were recovered, two from ground beef and two fromgrinding-machines; all harbored the stx 2 gene and were negative for the eae gene. All E. coli isolatesincluding the four STEC were screened for antibiotic resistance. High levels of resistance against severalantimicrobial agents were detected; those most commonly observed were to tetracycline (76.6%), amoxicillin(64.1%) and cephalothin (58.8%). Such high levels of antimicrobial resistance highlight the need for a morerational use of these agents in cattle.
Key words: Escherichia coli, STEC, stx 2, antibiotic resistance, ground beef, cattle
INTRODUCTION
1 (Stx 1) and Shiga Toxin 2 (Stx 2). Although different proteins,encoded by different genes (stx 1 and stx 2, respectively), they Shiga toxigenic Escherichia coli (STEC) has been have similar biological activities (18). Strains possessing stx 2 implicated as the cause of several human diseases including are potentially more virulent than those carrying stx 1 or even mild or severe bloody diarrhea (hemorrhagic colitis), hemolytic strains carrying both stx 1 and stx 2 (3,16). Another virulence syndrome (HUS) and renal failure (10,22). The STEC strain most factor, intimin (eae) is encoded in a pathogenicity island called frequently associated with clinical disease in the United States LEE (locus of enterocyte effacement). LEE-encoded genes and Europe is the serotype O157:H7 (4,16). However, several appear to enhance STEC virulence and have been associated other serotypes have been associated with severe disease outbreaks, and in some countries they have been isolated from Antimicrobial agents for therapy or prophylaxis aimed at clinical cases more often than O157 (13,16). Cattle is considered animal growth promotion, have favored propagation of resistant the primary reservoir of both O157 and non-O157 STEC bacteria bacteria (27). Intestinal resistant bacteria due to fecal (2). Several human infections have been linked to STEC in contamination during slaughtering may be transferred to meat undercooked ground beef (10). During processing, fecal products (6). If subsequently transmitted to human food (27), contamination of the carcass or transfer of bacteria found on the animal hide to the carcass could facilitate transmission of This study is aimed at the determination of STEC occurrence in ground beef and the characterization of antimicrobial Pathogenic STEC strains known to produce one or two toxins resistance of E. coli isolated from ground beef, meat-grinding resembling those from Shigella dysenteriae (19), are Shiga Toxin machines and hands of meat manipulators.
*Corresponding Author. Mailing address: Faculdade de Odontologia de Ribeirão Preto, Departamento de Morfologia, Estomatologia e Fisiologia, FORP- Via do Café S/N, Campus USP, 14040-904 Ribeirão Preto, SP, Brasil. Tel.: +55-16-36024101 Fax: +55-16-36330999. E-mail: jmmarin@forp.usp.br MATERIALS AND METHODS
Laboratory Standards (17), using commercial disks (Cefar, SãoPaulo, Brazil), loaded as follows: nalidixic acid (30 µg), amikacin Sampling
(30 µg), amoxicillin (10 µg), amoxicillin-clavulanic acid (30 µg), Meat samples and swabs from grinding-machines and hands ampicillin (10 µg), cephalothin (30 µg), ceftriaxone (30 µg), of meat manipulators were collected on separate occasions, ciprofloxacin (5 µg), cotrimoxazole (25 µg), streptomycin (10 over a 10-month period (March 2004 to January 2005), from µg), gentamicin (10 µg) and tetracycline (30 µg). E. coli reference twenty-three butcheries in Taquaritinga, a city in the northwest strains ATCC 25922 and ATCC 35218 were used for strain quality of the State of São Paulo. Samples were kept on ice between collection and testing. On the same day of collection, 25gsamples of ground meat were homogenized by hand in 225 ml of sterile 0.1% (wt/vol) peptone water (Oxoid Ltd, Basingstoke,Hampshire, UK), in a stomacher bag, for 5-10 min. Swabs from Among 287 E. coli isolates submitted to PCR to detect stx 1, machines or hands of operators were manually mixed with 10 ml stx 2 and eae genes, four STEC strains were isolated. Two of of 0.1% peptone water. One ml samples from each suspension them were from the same butchery (ground beef and grinding- were diluted in 9 ml of lauryl sodium sulfate broth (Difco machine) and the remaining two (one from ground beef and one Laboratories, Detroit, USA) and incubated for 24-48h at 35ºC.
from a grinding-machine) were from two different stores (Table One hundred µl samples from tubes showing bacterial growth 1). All isolates carried the stx 2 gene and were negative for the were added to 5 ml of Brilliant Green broth (Difco) or EC broth (Difco) and incubated as above. Ten µl of the broths positive Antibiotic resistance patterns of the isolates (n=287) are for coliform growth were plated on Eosin Metylene Blue (EMB- presented in Table 2. Isolates presenting intermediary resistance Difco) agar and incubated for 24h at 35°C. At least five colonies were also classified as resistant. The most frequent resistances were taken from the EMB plates for further identification (7).
were to tetracycline, amoxicillin, cephalothin and streptomycin.
Resistances to amoxicillin + clavulanic acid and ceftriaxone were Determination of stx genes
rare. Thirty (10.4%) isolates were sensitive to all antibiotics E. coli isolates were grown overnight in nutrient broth (Sigma Chemical Co, St Louis, USA) at 37ºC, and tested for stx Resistance to at least three drugs was found in 67.0% of the genes (stx 1 and stx 2) using the polymerase chain reaction isolates and 22.0% were resistant to more than 6 antibiotics.
(PCR) protocol of Orden et al. (20). DNA templates were The resistance patterns of the STEC isolates are presented in prepared by pelleting 1 ml of each culture by centrifugation Table 3. One of the isolates (strain 530) was resistant to 7 (12000xg), ressuspension in 250µl of sterile distilled water and antibiotics; another (strain 426) was susceptible to all drugs.
boiling for 10 min. After centrifugation, supernatants were usedfor PCR in an Eppendorf Mastercycler (Eppendorf AG,Hamburg, Germany). The amplified DNA products were Table 1. Isolation of STEC from butcheries in Taquaritinga city,
separated by electrophoresis on 1.5% agarose gel, stained with ethidium bromide and detected under ultraviolet light.
Reference E. coli strains used as controls were EDL 933 (O157:H7, stx1, stx 2, eae) and DH5α (negative control).
Characterisation of isolates
Isolates were confirmed as stx+ and tested for accessory virulence marker (eae) using the PCR protocol of China et al. (5).
O157 latex agglutination
DISCUSSION
STEC isolates were serotyped for the O serotype O157 using the O157 Latex Agglutination test kit (Oxoid, STEC strains are part of the microbiota of the gastrointestinal Basingstoke, Hampshire, UK). The EDL 933 strain was used tract of cattle raised for meat consumption. Transfer of fecal as a positive control. Strains negative for agglutination were material to carcasses at slaughter leads to potential contamination of raw meat (6), as also does cross-contaminationof meat and processing equipment (1).
Antibiotic susceptibility tests
All STEC isolated in this study carried the stx 2 gene, in STEC and non-STEC isolates were submitted to the disk agreement with Brazilian (11,14) and Argentinean reports (21) diffusion method according to National Committee for Clinical showing that stx 2 is more frequent than stx 1 in cattle. Absence Table 2. Antibiotic resistance of 287 E. coli isolates recovered
from a meat-grinding machine. This is a matter of concern from ground beef, grinding-machines and hands of meat because unsatisfactory cleaning of meat grinders have been manipulators in 23 butcheries in a Taquaritinga city, State of proven to be a source of contamination during processing (1,8).
Warriner et al. (26) demonstrated that the same clone of E. coliwas isolated from a pork carcass and from the equipment used The emergence and dissemination of antimicrobial resistance among E. coli, especially STEC strains from cattle, may have potentially negative clinical implications for humans. Thus continued surveillance of emerging antimicrobial resistance among zoonotic food-borne pathogens, including STEC is required to ensure public health protection. Although STEC O157: H7 had been considered sensitive to many antibiotics (24), recent results demonstrated resistance of STEC O157:H7, and in special the non-O157 STEC strains (9,12). Results of antimicrobial susceptibility testing (Table 2) showed high resistance to tetracycline, cephalothin, amoxicillin and streptomycin. These findings agree with data from previous studies showing that resistance is common among strains isolated from food, animals and meat (23,25).
Despite of low number of non-O157 STEC strains isolated in *Isolates presenting full and intermediary resistance were combined the present study (n = 4) some of these strains (Table 3) were resistant to four or more antibiotics, in agreement with the recentreport of Mora et al. (15), who described that among 581 non-O157 STEC strains, 239 (41.0%) were resistant to at least one of Table 3. Resistance patterns of the four STEC isolates recovered
the antibiotics tested. These authors detected multiple resistance in 71 strains, which were resistant to five or more Antimicrobial-resistant bacteria in food may be a source of resistance genes transferable to human intestinal microbiota (28). Control strategies should therefore be introduced by veterinary authorities to safeguard public health.
*-amox- amoxicillin; nalid- nalidixic acid; ceph- cephalothin; tet- ACKNOWLEDGEMENTS
tetracycline; cipro- ciprofloxacin; amik- amikacin; amp- ampicillin.
**- Susceptible to all tested antibiotics.
The authors thank Tania Marques da Silva for technical assistance. Financial support was from FAPESP.
or rarity of the eae gene in STEC isolates coincides with earlier reports (11,14). Absence of serotype O157: H7 in STEC isolatesis not unexpected; it is extremely rare (0.6%) in Brazilian cattle Isolamento de Escherichia coli Shiga toxigênica em
(11). Interestingly, two recovered isolates did not express stx açougues na cidade de Taquaritinga, Estado de São
Paulo, Brasil
Hussein and Bollinger (10), in a review of the prevalence of STEC in beef, reported prevalence of non-O157 STEC ranging Escherichia coli Shiga toxigênica (STEC) tem sido between 1.7% and 58.0% in samples from packing plants and responsabilizada como o agente etiológico de diversas doenças between 3.0% and 62.5% in supermarket samples; the USA, nos seres humanos. Neste estudo foram analisadas amostras England, Canada and India showed the highest frequencies on provenientes de 23 açougues (carne moída, moedor de carne e isolation. In the present study, we found 1.3%, a value lower mãos de manipuladores) visando o isolamento de cepas de E. coli utilizando os métodos microbiológicos tradicionais. Um Two of the STEC isolates, strains 393 and 426, were recovered total de 287 cepas de E. coli, isoladas destas amostras, foram from the same butchery, one from ground beef and the other submetidas a reação em cadeia da polimerase para a detecção dos genes stx 1, stx 2 e eae. Foram identificadas 4 cepas STEC, 12. Khan, A.; Das, S.C.; Ramamurthy, T.; Sikdar, A.; Khanam, J.; 2 provenientes de carne moída e 2 provenientes do moedor de Yamasaki, S.; Takeda, Y.; Balakrish Nair, G. (2002). Antibioticresistance, virulence gene and molecular profiles of Shiga Toxin- carne, todas as cepas apresentando o gene stx 2 e negativas producing Escherichia coli isolates from diverse sources in Calcutta, para a presença do gene eae. Todas as cepas de E. coli, incluindo India. J. Clin. Microbiol., 40, 2009-2015.
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