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.
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
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
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
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: email@example.com
MATERIALS AND METHODS
Laboratory Standards (17), using commercial disks (Cefar, SãoPaulo, Brazil), loaded as follows: nalidixic acid (30 µg), amikacin
(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
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
machines or hands of operators were manually mixed with 10 ml
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
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
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
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
) 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.
O157 latex agglutination
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
Antibiotic resistance of 287 E. coli
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. coli
was 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
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-
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
Hussein and Bollinger (10), in a review of the prevalence of
STEC in beef, reported prevalence of non-O157 STEC ranging
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.
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
2 e eae
. Foram identificadas 4 cepas STEC,
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