OCCURRENCE OF SHIGA TOXINPRODUCING Escherichia coli NONO157 IN READYTOEAT FOOD FROM SUPERMARKETS IN

1
OCCURRENCE OF SHIGA TOXIN-PRODUCING Escherichia
coli NON-O157 IN READY-TO-EAT FOOD FROM
SUPERMARKETS IN ARGENTINA

• C. BALAGUÉ, L. FERNÁNDEZ, A.L. REDOLFI, V. AQUILI
• Faculty of Biochemical and Pharmaceutical
  Sciences, National University of Rosario,
  Suipacha 531, 2000 Rosario, Argentina
•  
• P. VOLTATTORNI, C. HOFER, G. EBNER, S. DUEÑAS, L.
  BAITA
• Rosario Food Institute, Lima 865, 2000 Rosario,
  Argentina
•  
• A. KHAN, C. CERNIGLIA
• Division of Microbiology, U. S. Food and Drug
  Administration, NCTR, 3900 NCTR Road., Jefferson,
  AR 72079, USA
•  

2
Abstract (1)

• Between June 2000 and December 2001, 500 food
  samples were collected from supermarkets and
  shops selling ready-to-eat food in Rosario,
  Argentina, and examined for Escherichia coli.
  Forty-nine E. coli isolates from food samples
  were further characterized for virulence genes by
  multiplex polymerase chain reaction (PCR)
  targeting the stx1, stx2, stx2e, eaeA, CNF1,
  CNF2, Einv, LTI, STI, and STII genes in four
  groups. Out of forty-nine E. coli isolates
  screened by multiplex PCR, ten showed the Shiga
  toxin stx1and stx2 genes and none showed the
  other genes. The Shiga toxin positive E. coli
  strains (STEC) were isolated from soft and
  cottage cheeses. These E. coli isolates were
  serogrouped and belonged to O18 (two strains),
  O8, O57w, O79, O44, and O128 three strains were
  untypeable. Pulsed-field gel electrophoresis
  (PFGE) with XbaI generated a unique profile for
  each, having ten to fifteen bands ranging from 50
  to 500 kb, except the strain ARG20 that generated
  small bands and was partly degraded. Analysis of
  the biotypes of STEC isolates showed variations
  and four strains (ARG5468, ARG5266, ARG2379, and
  ARG4823) revealed characteristics compatible with
  E. coli O157H7, namely that they do not produce
  ß-glucuronidase, or ferment sorbitol. Strain
  ARG4827 expressed three characteristics
  compatible with enteroinvasive E. coli (EIEC),
  was negative for sucrose, esculin, and salicin.
  Hemolysin and fimbriation tests showed expression
  of fimbriae and hemolysins in several isolates.
  Strain ARG20 expressed fim-P, ARG177 expressed
  fim-CFA, ARG5468 and ARG5266 expressed
  enterohemolysin and ARG2379 was positive for
  a-hemolysin.

3
Abstract (2)

• Results revealed that these shiga toxin-producing
  Escherichia coli non-O157 strains are potential
  foodborne pathogens and their presence in
  ready-to-eat food illustrates the need to keep a
  careful watch over where are pathogens coming
  from and then develop methods to control them.
• Acknowledgements
• This work was supported in part by the visiting
  Scientist Program at the National Center for
  Toxicological Research administered by the Oak
  Ridge Institute for Science and Education through
  an interagency agreement between the U. S.
  Department of Energy and the Food and Drug
  Administration.

4
Introduction (1)

• Escherichia coli is a predominant component of
  the intestinal flora of humans and other mammals.
  Yet, several E. coli strains represent primary
  pathogens with an enhanced potential to cause
  diseases, specifically diarrhea and urinary tract
  infection (9, 17, 33). Thus, a great diversity of
  strains belonging to many different serotypes can
  be isolated from the environment and may
  contaminate food (8, 12, 14, 23, 24). These
  strains possess many virulence genes located in
  the chromosome or in mobile elements like
  plasmids, phages or transposons (5, 19, 20. 31).

5
Introduction (2)

• Uropathogenic strains (UPEC) contains the genes
  encoding
• RTX toxins
  designated ?-hemolysin
• Cytotoxic
  necrotizing factor (CNF)
• Adherent P
  fimbriae (19, 21)
•  
• Enteropathogenic E. coli (EPEC), contains the
  locus of enterocyte effacement with
• eaeA gene,
  encoding the bacterial outer-membrane protein,
  intimina (4).
  

6
Introduction (3)

• Enterotoxigenic E. coli (ETEC) contains
• Plasmid
  encoded CFA (colonization factor antigen)
  fimbriae
• Heat labile
  (LT) and/or heat -stable (ST) enterotoxins (9).
• Enteroinvasive E. coli (EIEC) contains
• Protein Einv and
  multiplies within the epithelial cells lining the
  colon (18).

7
Introduction (4)

• An emergent pathogen, Shiga toxin-producing E.
  coli (STEC) of the serogroup O157, is designated
  as enterohemorrhagic E. coli (EHEC) and has been
  responsible for many outbreaks of hemorrhagic
  colitis and hemolytic uremic syndrome (14, 21).
  Many STEC possess the eaeA chromosomal gene,
  Shiga toxin 1 and/or Shiga toxin 2 (stx1, stx2)
  genes encoded on bacteriophages and a large
  plasmid that encodes the toxin enterohemolysin
  (13, 21, 26). Stx can damage endothelial cells
  as well as tubular cells, which may result in
  acute renal failure (33) and is regarded as the
  major virulence factor of STEC.

8
Introduction (5)

• In Argentina, where hemolytic uremic syndrome is
  endemic (32), the estimated annual incidence rate
  in children is 9.2 per 100,000. Several
  outbreaks caused by non-O157 STEC were described
  (14) although data implicating these STEC in
  some outbreaks were scanty and the source of
  infection was not always known.
• STEC non-O157 (O8, O18, O127) strains previously
  associated with enterohemorrhagic colitis and of
  bloody diarrhea (8, 25) were isolated from bovine
  fecal samples in Argentina (12). However,
  characteristics and virulence properties of
  non-O157 strains isolated from ready-to-eat food
  samples have not been previously reported in
  Argentina geographical area.
• The commonly used detection methods in food
  microbiology, involving culture on sorbitol
  MacConkey agar, often with an immunomagnetic
  separation step, are capable only of detecting E.
  coli O157 (28). Non-O157 STEC, as well as EPEC,
  EIEC, UPEC and ETEC, are often overlooked.

9
Objective

• To examine pathogenic E. coli in samples obtained
  from supermarkets and shops selling ready-to-eat
  food in the Rosario area. In addition, we
  studied the phenotypic and genotypic
  characteristics of the strains and the clonal
  relationship of the STEC isolates.

10
Materials and Methods (1)

• Bacterial strains. From June 2000 to December
  2001, 500 food samples obtained from supermarkets
  and shops selling ready-to-eat food in the
  Rosario, Argentina, were examined for E. coli.
  The samples were randomly collected by inspectors
  from the Rosario Food Institute by the
  recommended methodology of the International
  Commission of Microbiological Specifications for
  Food (ICMSF). E. coli strains were isolated and
  identified as described previously (23).

11
Materials and Methods (2)

• E. coli O157H7 ATCC43890 and 933J from the
  National Institute of Infectious Diseases
  (Argentina) which possess the Shiga toxin gene
  stx1 ATCC43889 and ATCC43895 from the American
  Type Culture Collection, which express the Shiga
  toxins genes stx2 and both stx1 and stx2,
  respectively, and the attaching-and-effacing
  lesion gene (eaeA) were used as positive
  controls. The strain ATCC43895 harbors the
  enterohemolysin gene encoded in a 60 Mda plasmid
  (27) and the stx2 phage, 933W (28).
  Enterotoxigenic E. coli ATCC35401, which
  expresses LTI and STI, and E. coli T149, which
  express fimbriae P and ?-hemolysin (1) were also
  used as positive controls. E. coli HB101 and
  ATCC98222 were used as negative controls.

12
Materials and Methods (3)

• Biochemical characterization. Typing was made
  with additional biochemical tests to identify
  EIEC and EHEC lysine decarboxylase, motility,
  sucrose, salicin, esculin, glycerol,
  ?-glucuronidase and sorbitol (7).
• Serogrouping. Serogrouping was done by Penn
  State University, E. coli Reference Center.
• Detection of adhesions. ETEC and UPEC fimbriae
  were identified as types CFA and P by
  mannose-resistant hemagglutination of bovine
  erythrocytes and human group A erythrocytes,
  respectively (3, 11, 17). Induction of fimbrial
  expression was tested by successive passages on
  culture media supplemented with casamino acids.

13
Materials and Methods (4)

• Detection of toxins UPEC ?-hemolysin. The
  strains were grown during 4 h in alkaline meat
  broth with 0.2 glucose. Dilutions of the
  supernatants were incubated during 4 h at 37 ºC
  with a suspension of sheep erythrocytes (5) in
  saline solution. The liberated hemoglobin was
  measured at 540 nm (30).
• EHEC. The production of enterohemolysin was
  detected using tryptic soy agar base with 5 of
  washed sheep erythrocytes and supplemented with
  10 mM CaCl 2. The blood agar plates were
  examined after 4 and 24 h incubation at 37?C.
  Hemolysis on the washed blood agar plates after
  24 h but not after 4 h was considered to be
  enterohemolysin (2). Tests of hemolysis
  induction were made by successive passages on
  culture media supplemented with blood.

14
Materials and Methods (5)

• Multiplex PCR. The chromosomal DNA of the
  strains was extracted with the Qiagen genomic DNA
  extraction kit (Qiagen, Inc., Valencia, CA, USA)
  according to the manufacturers instructions.
  Specific primers were used to amplify the genes
  that encode specific virulence factors from the
  different virotypes, such as Stx 1, Stx 2, Stx
  2e, eaeA, CNF 1, CNF 2, LT I, ST I, ST II and
  Einv, according to the multiplex PCR conditions
  described by Pass et al (2000).

15
Materials and Methods (6)

• Pulsed- field gel electrophoresis (PFGE). The
  isolate fingerprints generated in this study were
  based on PFGE separation of XbaI-digested genomic
  DNA (6) this is the method used by members of
  PulseNet. Briefly, bacterial cultures were grown
  on tryptic soy agar at 37?C, and after 24 h a
  suspension of each culture in 100 mM Tris/100 mM
  EDTA was made. Agarose plugs were prepared by
  mixing equal volumes of the bacterial suspension
  with 1 agarose in TE (10mM Tris/HCl, 1 mM EDTA,
  pH 8.0) plus 0.5 mg proteinase K (Gibco-BRL) and
  casting into molds (Bio-Rad Laboratories).
  Bacterial cells in the agarose plugs were lysed
  by treatment with lysis solution containing 0.1
  mg/ml proteinase K in a solution of 50 mM Tris,
  50 mM EDTA and 1 N-lauroyl sarcosine for 2 h at
  55?C. Agarose plugs were washed twice in sterile
  distilled water and four times in TE at 50?C for
  a minimum of 20 min for each wash. A section of
  each plug was placed into 200 µl of enzyme buffer
  containing 50 U of the restriction enzyme XbaI
  for 6 h at 37?C. Restriction fragments were
  resolved in a 1 agarose gel in 0.5 X TBE
  buffer, using a contour-clamped homogeneous
  electric field apparatus (CHEF-DR II, Bio-Rad
  Laboratories).

16
Results (1)

• From the 500 food samples analysed, most of them
  were negative in the microbiological test, some
  of them were positive for another lactosa
  fermenting or non-fermenting microorganisms and
  48 of them were Escherichia coli positive.
  Forty-nine strains of E. coli were isolated from
  48 samples from soft cheese, cottage cheese,
  salads with cream or mayonnaise, food with
  sauces, and sandwiches. These isolates were
  assayed for four multiplex PCRs to detect the
  presence of 10 virulence genes. The following
  combinations of primers gave adequate
  amplification of the respective target genes Stx
  1, Stx 2, Stx 2e and eae A CNF 1 CNF 2 and
  Einv and LT I, ST I and ST II. Figure 1 shows
  the presence of the amplified product profiles
  for ten (20.4 ) of the 49 isolates and the
  reference E. coli strains (positive and negative
  controls), after agarose gel electrophoresis. PCR
  primers in set stx1, stx2, stx2e and eaeA
  amplified fragments of DNA of the predicted size
  for stx1 and stx2 (22). The two observed PCR
  products elucidate the presence of both Shiga
  toxin genes in the isolates, but the absence of
  eaeA and stx2e genes. The occurrence was
  particularly associated with samples from soft
  and cottage cheese (70 of the ten STEC
  strains). The other 3 sets of primers assayed
  revealed negative results (Table1). When the
  other 39 isolates were tested with the four
  multiplex PCR, results did not detect any of the
  10 major virulence genes.

17
Results (2)

• Figure 1. Agarose gel electrophoresis of
  multiplex PCR products of isolates STEC. Lane 1,
  molecular weight standard (100-bp) lane 2, ATCC
  43890 lane 3, ATCC 43889 lane 4, ATCC 43895
  lane 5, E. coli 933J lane 6, ATCC 98222 lane 7,
  ARG 4828 lane 8, ARG 177 lane 9, ARG 20 lane
  10, ARG 4827 lane 11, ARG 5468 lane 12, ARG
  5266 lane 13, ARG 2379 lane 14, ARG 4823 lane
  15, ARG 4627 lane 16, ARG 4824.
• Expected sizes for the PCR products are stx1,
  121 stx2, 102 eaeA, 241.

18
Table1. Genotypic characterization of STEC
isolates from different food samples obtained in
Rosario, Argentina PFGE pulsed- field gel
electrophoresis, PCR polimerasa chain reaction
19
Table1. Genotypic characterization of STEC
isolates from different food samples obtained in
Rosario, Argentina(cont.) PFGE pulsed- field
gel electrophoresis, PCR polimerasa chain
reaction
20
Results (3)

• The STEC isolates were serogroup non-O157 as
  described in Table 2, two of them were O18,
  others were non-typeable and the rest represented
  O8, O44, O57w, O79 and O128. With the objective
  of characterizing the different strains isolated
  from food for biotypes, 30 biochemical tests were
  performed for the 49 E. coli isolates. For the
  following tests, the same results were observed
  in all the strains citrate, urea, TSI (hydrogen
  sulfide production on triple sugar iron),
  arginine dihydrolase, Voges Proskauer, methyl
  red, phenylalanine deaminase, D-xylose,
  D-mannitol and glucose. Another 20 biochemical
  tests revealed variable results for the different
  isolates.

21
Results (4)

• Biotype analysis of the STEC isolates also showed
  a great variety between strains. Four strains (40
  ) revealed two characteristics compatible with
  E. coli O157H7 ARG5468, ARG5266, ARG2379 and
  ARG4823 did not produce ?-glucuronidase and did
  not ferment sorbitol. One of the strains
  (ARG4827) expresses three characteristics
  associated with EIEC sucrose, esculin and
  salicin were negative (Table 2). These
  characteristics were also present in some of the
  39 non-STEC isolates. Three strains were
  ?-glucuronidase and sorbitol negative, and
  another five had characteristics associated with
  EIEC. According to the biochemical variety of
  the strains, the results of subtyping with PFGE
  demonstrated differences in the pattern profiles
  of the ten isolates. Moreover, all of them were
  different from the control E. coli strains. PFGE
  produced 10 to 15 fragments ranging in size from
  approximately 50 to 500 kb with the only
  exception for strain ARG20 that had a mucoid
  colony and was partly degraded (Figure 2).

22
Results (5)

• Because different E. coli virotypes that are
  producers of diarrhea express an important
  diversity of virulence factors, we determined the
  factors more frequently associated with each one
  of them expression of fimbriae and hemolysin
  (Table 2). Also considering that the expression
  of toxins and fimbriae can be inhibited outside
  of the animal host, tests of induction were made
  by means of successive passages on culture media
  supplemented with blood or casamino acids.
  However, only one of the STEC strains (10)
  revealed expression of fimbriae type P, CFA or
  ?-hemolysin. These results were similar to those
  obtained with the 39 non-STEC isolates (7.7 ,
  7.7 and 5.1 , respectively).
• Two other STEC strains expressed
  enterohemolysin (20), a percentage that was
  almost eight times higher than the percentage
  obtained with the other 39 isolates (2.6 ).
  Enterohemolysin was characterized by the
  production of small zones of hemolysis after 24 h
  incubation on blood agar containing washed
  erythrocytes. In contrast, E. coli ?- hemolysin
  released hemoglobin from suspended sheep
  erythrocytes and large clear zones of hemolysis
  were apparent after 4 h incubation with washed
  and unwashed erythrocytes.

23
Results (6)
 

• Figure 2. XbaI-digested PFGE patterns of STEC
  strains. Lane 1, low-range PFGE marker lane 2,
  ATCC 43890 lane 3, ATCC 35401 lane 4, ATCC
  43889 lane 5, ATCC 43895 lane 6, ARG 4828 lane
  7, ARG 177 lane 8, ARG 20 lane 9, ARG 4827
  lane 10, ARG5468 lane 11, ARG 5266 lane 12, ARG
  2379 lane 13, ARG 4823 lane 14, ARG 4627 lane
  15, ARG 4824.

24
Table 2. Phenotypic characterization of STEC
isolates from food samples obtained in Rosario,
Argentinasor sorbitol, mug ?-glucuronidase,
esc esculin, lis lysine, sac sucrose, sal
salicin, hem hemolisin, fim fimbriae
25
Table 2. Phenotypic characterization of STEC
isolates from food samples obtained in Rosario,
Argentina(cont.) sor sorbitol, mug
?-glucuronidase, esc esculin, lis lysine, sac
sucrose, sal salicin, hem hemolisin, fim
fimbriae
26
Discussion (1)

• In E. coli, pathogenicity is a complex
  multifactorial mechanism involving a large number
  of virulence factors, which vary according to the
  virotype. They include fimbrial attachment
  functions, invasins and many different toxins as
  well as secretion systems, which export toxins
  and other virulence factors (9, 10, 15, 16). The
  genome of E. coli is of high plasticity, allowing
  it to gain and lose virulence genes encoded in
  mobile elements like plasmids and phages. The
  combination of DNA elements encoding different
  virulence determinants might lead to the
  emergence of new pathogenic types. A general
  assessment of the genetic potential of virulence
  of E. coli isolates has a particular interest for
  strains isolated from ready-to-eat food, which
  may represent an important reservoir of virulence
  genes.

27
Discussion (2)

• Our results did not reveal any strains belonging
  to the virotypes UPEC, EPEC, ETEC, and EIEC.
  Since merely isolated virulence factors could be
  detected phenotypically as ?-hemolysin and
  fimbriae type CFA and P, it is not possible to
  conclude the presence of pathogenicity islands in
  our isolates.

28
Discussion (3)

• STEC are now recognized as an important group of
  bacterial enteropathogens (21) however, only
  O157H7 is the best known to both microbiologists
  and the general public. The plethora of STEC
  strains that are non-O157 have generally been
  poorly characterized and the incidence of
  virulence properties in these strains is largely
  unknown. Altogether, about 250 non-O157 STEC
  serotypes have been reported and more than 100 of
  them have been associated with human illness.
  Some estimates suggest that, in the USA, 25 of
  hemolytic uremic syndrome cases are caused by
  non-O157 STEC (14, 16). The relative isolation
  rates of non-O157 STEC vary from study to study
  and are influenced both by geographical area and
  detection methods.

29
Discussion (4)

• For rapid and sensitive detection of STEC
  non-O157 strains from clinical samples, PCR has
  proven to be of great diagnostic value in the
  detection of stx genes (10). In the
  epidemiological research, pulsed-field gel
  electrophoresis (PFGE) has been discriminative in
  the molecular comparison of STEC strains
  independent of the serotype (24, 34). In the
  present study, we utilized multiplex PCR, O
  serogrouping, biochemical tests, hemolysin and
  fimbrial detection and we used PFGE for the
  characterization of all E. coli non-O157 strains
  isolated from ready-to-eat food in the Food
  Institute of Rosario, Argentina. All samples
  analyzed complemented the microbiological quality
  control for coliform counts, but we analyzed them
  considering the importance of low infectious
  doses estimated in STEC (29).

30
Discussion (5)

• In routine food analysis, there is no definitive
  biochemical characteristic, such as sorbitol
  fermentation, which would distinguish STEC
  strains belonging to serogroups other than O157.
  However, in clinical samples, a significant
  proportion of non-O157 STEC produce
  enterohemolysin (10) and this phenotypic
  characteristic was used to facilitate the
  isolation of sorbitol-positive non-O157 STEC
  strains. In our study, phenotyping with several
  tests was unhelpful to facilitate the isolation
  of these strains in food samples, because of the
  great variety of the results. Despite the fact
  that enterohemolysin was expressed in STEC
  strains, we could only detect 20 of the isolates
  by this phenotypic method and another 20 were
  distinguished by the ?-glucuronidase and sorbitol
  negative tests. Only multiplex PCR was able to
  detect all the non-O157 STEC. Thus, PCR
  comprising the set of known major virulence genes
  would be of great help to detect newly emerging
  clones of STEC.

31
Discussion (6)

• In this study we found some serogroups frequently
  associated with enterohemorrhagic colitis, like
  O18 and O8. STEC belonging to these serogroups
  also produced Shiga toxins and were recently
  recovered from cases of diarrheal illness and
  from fecal samples of healthy steers in Argentina
  (8, 12).
• Our findings imply an important human exposure to
  non-O157 STEC from ready-to-eat food. Bacterial
  characterization to determine the serotype and
  biotype is part of the quality assessment of food
  and water (14). Our results demonstrated that
  PCR for Shiga toxins is highly specific and must
  be used to investigate populations of STEC.
  These strains are potential foodborne pathogens
  and their presence in ready-to-eat food
  illustrates the need to keep a careful watch over
  which pathogens are causing human disease,
  determine where they are coming from and then
  develop methods to control them.

32
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