Antibiotic susceptibility pattern of Escherichia coli strains with verocytotoxic E. coli-associated virulence factors from food and animal faeces

FOOD MICROBIOLOGY Food Microbiology 20 (2003) 27–33

www.elsevier.nl/locate/jnlabr/yfmic

Antibiotic susceptibility pattern of Escherichia coli strains with verocytotoxic E. coli-associated virulence factors from food and animal faeces Gunter . Kleina,*, Michael Bulte . b a

Bundesinstitut fur (BgVV), Diedersdorfer Weg 1, D-12277 Berlin, Germany . gesundheitlichen Verbraucherschutz und Veterinarmedizin . b Institut fur Nahrungsmittelkunde, Justus-Liebig-Universitat, . Tierarztliche . . 35392 Gießen, Germany Received 30 May 2002; accepted 7 August 2002

Abstract There is limited information available on the antibiotic susceptibility patterns of enterohemorrhagic Escherichia coli (EHEC) or Verocytotoxin-producing E. coli (VTEC). Therefore in vitro antibiotic activities of 13 antibiotic substances against 60 E. coli isolates (from food or animal and one human faecal isolate) with VTEC-associated virulence factors were determined. The isolates harboured at least one of the following virulence factors: vtx1 and/or vtx2 gene, eae gene (EHEC or EPEC) or EHEC hly A gene. There was no relationship between serotype, virulence factor and resistance pattern. All strains were susceptible to quinolones, gentamicin, trimethoprim/sulfamethoxazole and nitrofurantoin. Resistant isolates were observed for cephalothin, tetracycline and in one case for cefazolin. Minimal inhibition concentrations at which 90% of the strains were inhibited were 8 mg ml
1 for ampicillin, 8/4 mg ml
1 for ampicillin/sulbactam, >16 mg ml
1 for cephalothin, 8 mg ml
1 for tetracycline and 8 mg ml
1 for chloramphenicol. No multi-drug-resistance was observed. Cephalothin and tetracycline show the potential for an increased resistance rate as a high number of isolates was intermediate resistant. The resistance profile of E. coli with VTEC-AVF investigated in this study reveals a high percentage of susceptible isolates compared to E. coli isolates in general and is similar to profiles published for EHEC or VTEC strains. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Escherichia coli; VTEC; EHEC; Antibiotic susceptibility; Food; Animal faeces

1. Introduction Within the European Union (EU) there exist efforts to establish an antibiotic resistance monitoring programme for the most important micro-organisms derived from animals. The revision of the EU guideline for zoonoses focuses on three categories: zoonotic bacteria (Salmonella and Campylobacter), commensal bacteria (E. coli and enterococci) and animal pathogens (enteropathogenic E. coli, Streptococcus spp., etc.) (Caprioli et al., 2000). So far no regular EU wide monitoring system has been established (OIE, 2001). On the other hand, a great variety of studies has been performed to investigate the antibiotic resistance of the most relevant bacterial species of veterinary and human *Corresponding author. Tel.: +49-1888412-2107; fax: +49-1884122951. E-mail address: [email protected] (G. Klein).

importance. Especially E. coli has been tested very intensely from many different sources (e.g. animal and food) (Lehn et al., 1996; Trolldenier, 1996; Altieri and Massa, 1999; Mathew et al., 1999). For monitoring programmes EHEC, VTEC or E. coli with verocytotoxigenic E. coli-associated virulence factors (VTECAVF), such as eae gene or hly gene, are not suitable as other infectious agents are of higher importance, like Salmonella and Campylobacter (Caprioli et al., 2000). Nevertheless, investigations are currently needed in order to gain information on the development of antibiotic resistance in these potentially pathogenic agents entering the food chain. In Europe only a limited number of studies concerning the antibiotic resistance of E. coli with VTEC-AVF has been performed (Stephan and Kuhn, . 1999; Threlfall et al., 2000; Stephan and Schumacher 2001). Some studies focussed on human clinical isolates (Schmidt et al., 1994), others investigated a broad range of isolates

0740-0020/03/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 7 4 0 - 0 0 2 0 ( 0 2 ) 0 0 1 0 6 - 5

28

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33

from animals, food and asymptomatic human carriers and different serotypes (Farina et al, 1996; Stephan and Schumacher, 2001). Data from the United States of America have been collected for E. coli O157:H7 by the Centers for Disease Control and Prevention (CDC) from 1996 to 1999 (CDC, 1997, 1998, 1999) and earlier studies (Kim et al., 1994; Meng et al., 1998). Specific attention was given to the antibiotic susceptibility of eae- and eae/stx-positive E. coli strains in healthy calves and calves with diarrhoea by a study by Holland et al., (1999). In Canada, the antibiotic resistance of VTEC isolated from pigs and humans was investigated (desRosiers et al., 2001). Therefore, a broader range of E. coli with VTEC-AVF such as vtx1/2 and/or eae and/ or EHEC hly A gene needs to be tested for their antibiotic resistance patterns. The resistance patterns of E. coli with VTEC-AVF are not primarily of interest in relation to therapeutic treatment (Beutin et al., 2000). Moreover, also for clinical relevant EHEC strains the use of antibiotics in the therapy of these infections is questionable (Wong et al., 2000; Gill and Hamer, 2001). Most EHEC cases are treated without the use of antibiotics. However, the knowledge of the resistance patterns is of crucial importance for an overview of the distribution of antibiotic resistances in food of animal origin and the contribution by E. coli with VTEC-AVF as a potential reservoir for EHEC strains. EHEC strains have been considered so far as susceptible to most antibiotics, but resistance has been observed to fluoroquinolones, tetracycline, sulfonamides and beta-lactams (Doyle et al., 1997). Therefore the aim of this study was to collect information on the prevalence of resistance in E. coli with VTEC-AFV in comparison to published resistance rates of E. coli without virulence factors and VTEC strains.

2. Material and methods In this study 60 E. coli strains which harboured at least one of the following VTEC-AVF were included. The factors were: Verotoxin genes (vtx1, vtx2), eae gene (EHEC or EPEC) and/or EHEC hly A gene. The strains were isolated between 1987 and 1998 from different sources. The isolation of these strains has been described previously (Montenegro et al., 1990; Bulte . et al., 1996; Hallmann et al., 1997). An overview on the sources of the isolates is given in Table 1. The strains were isolated from minced meat (beef) (n ¼ 30), raw sausages and meat products (9), sheep (faeces, meat) (15), cattle faeces (5) and human faeces (1). The E. coli strains from meat and meat products were isolated from different batches and production days. Serotyping was performed by Dr. Aleksic (Hygiene Institute, Hamburg) and has been described previously (Montenegro et al., 1990;

Table 1 Origin and virulence factors of the isolates Origin

Virulence gene vtx1

Minced meat/cattle Faeces/cattle Meat/lamb Faeces/sheep Sausage Human a b

b

0/30 2/5 5/5 10/10 4/9 0/1

vtx2

eaea

EHEC hly A

3/30 4/5 0/5 2/10 4/9 1/1

28/30 1/5 5/5 9/9 6/9 1/1

5/17 5/5 5/5 9/9 6/9 1/1

EHEC/EPEC eae. n positive/n tested.

Bulte . et al., 1996; Hallmann et al., 1997). The specific distribution of VTEC-AVF for all strains is demonstrated in Table 2. PCR detection was confirmed by methods and primers described previously (Bulte . et al., 1996; Hallmann et al., 1997). Briefly, the PCR protocol was as follows: DNA isolation was performed by the boiling method. DNA concentration was measured by using a DNA Fluorometer (Model TKO 100, Hoefer Scientific Instruments, San Francisco, California, USA). Oligonucleotides that were used for PCR amplification were synthesized by MWG Biotech GmbH (Ebersberg, Germany) and the following primer pairs were used: KS7/KS8 for vtx1 (Schmidt et al., 1994), GK5/GK6 for vtx2 (Schmidt et al., 1994), SK1/SK2 for the eae gene (Schmidt et al., 1994), LP1/LP3 for complete eae gene (Schmidt et al., 1994) and hlyA1/hlyA4 for the EHEChemolysin gene (Schmidt et al., 1995). The reactions were performed in a total volume of 25 ml with 2.5 ml of DNA in each test. Amplification was performed with PrimeZyme DNA Polymerase Kit in a Trio-Thermo. block cycler (both Biometra, Gottingen, Germany). The reaction mix was overlaid with mineral oil (Sigma, Deisenhofen, Germany). The cycle parameters were as follows: for primer KS7/KS8 and GK5/GK6: denaturing step: 941C, 30 s; annealing step: 521C, 60 s; extension step: 721C, 40 s; 30 cycles; for primer SK1/SK2: 941C, 30 s (the first denaturing step was extended by 5 min); 521C, 60 s; 721C, 45 s; 30 cycles; for primer LP1/LP3: first denaturing step: 941C, 5 min; 3 cycles as follows: 941C, 45 s; 431C, 60 s; 721C, 2.5 min; 27 cycles as follows: 941C, 45 s; 521C, 60 s; 721C, 2.5 min; for primer hlyA1/ hlyA4: 941C, 30 s (the first denaturing step was extended by 5 min); 571C, 90 s; 721C, 90 s; 30 cycles. DNA from E. coli 4884 (vtx, eae and EHEC hly A positive) and E. coli 2391 (vtx, eae and EHEC hly A negative) (both from the strain collection of M. Bulte, . Justus-Liebig-University, Gie
en, Germany) were used as controls and aqua bidestillata was used without DNA for contamination control. The PCR fragments were separated by submarine agarose gel electrophoresis

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33

29

Table 2 Characterization of strains Straina

Serotype

Resistance patternb

PCR of virulence genec vtx1

vtx2

eae

EHEC eae

EHEC hly A

d

MC173 MC794 MC796 MC1449 MC1597 MC1708 MC1801 MC1836 MC1842 MC1843 MC1844 MC1845 MC1846 MC1847 MC1851 MC1852 MC1856 MC1857 MC1868 MC1870 MC1872 MC1875 MC1876 MC1885 MC1886 MC3106 MC3115 MC3174 MC3200 MC3336

O1:H2 O15:H17 ND O156:H47 O156:H47 O56:HO1:H2 O156:H47 ND ND ND ND ND ND ND ND O156:H47 ND ND ND ND ND ND O156:H47 ND O1:H2 O56:HO26:H11 O26:H11 O146:H57

Cep Cepd Chld Tetd Cepd Tetd Cepd Tetd Tetd Tetd Cepd Cepd Cepd Tet Cepd Cepd Cepd Tetd Cepd Cepd Tetd Cepd Cepd Cepd Cepd No Cepd AmSd Cepd Cepd Cepd No Tetd Cepd Tetd Chld Chld Cep Tetd

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

+ — — — — — — — — — — — — — — — — — — — — — — — — + + — — —

— + + + + + + + + + + + + + + + + + + + + + + + + — — + + +

+ — — + + — + + ND ND + ND ND ND ND ND + ND ND ND ND ND ND + + ND ND — — +

— — — — — — — — ND ND — ND ND ND ND ND — ND ND ND ND ND ND — — — — + + —

FC2324 FC2403 FC2430 FC2443 FC2481

O157:H7 O3:HO116:H21 O113:H21 O126:H21

Cepd Cepd No Cep No

— + + — —

+ — + + +

+ — — — —

+ ND ND ND ND

+ + + + +

ML261 ML262 ML264 ML265 ML268

O119:HO70:H11 O156:H25 O70:H11 O107:H11

Cepd Cepd Cepd Cepd Cepd

+ + + + +

— — — — —

+ + + + +

— — — — —

+ + + + +

FS139 FS140 FS141 FS142 FS143 FS172 FS199 FS201 FS207 FS208

ND O84:H31 On.t.:HO84:H31 ND O156:H26 O156:H25 O15:H+n.t. O156:HND

Cepd Cepd Cepd Cepd Cepd Cepd Cepd No Cepd Cepd

+ + + + + + + + + +

— — — — + + — — — —

+ + + + ND + + + + +

— — — — ND — — — — —

+ + + + ND + + + + +

S6097 S6102 S6204 S6235 S-CH1 S-CH2 S-CH3

O62:H32 O2:HO3:H2 O22:H7 O26:H11 O149:H12 O76:H32

Tet AmSd Ampd Cepd Chld Tet Chld Tet Tetd Cepd Cep Cef Ampd Cepd Tet

— — + — — + +

— — — — — + +

+ + — + + — +

+ + ND + — ND +

+ — — + + — +

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33

30 Table 2 (continued) Straina

S-E140 S-Y/552 H1

Serotype

O157:H7 O121:H10 O157:H7

Resistance patternb

No Cep No

PCR of virulence genec vtx1

vtx2

eae

EHEC eae

EHEC hly A

+ — —

+ + +

+ — +

+ ND +

+ — +

a

Origin: MC: minced meat/cattle; FC: faeces/cattle; ML: meat/lamb; FS: faeces/sheep; S: sausage; H: human. Resistance pattern (resistant or intermediate resistant): Cep: Cephalothin; Tet: Tetracycline; Chl: Chloramphenicol; AmS: Ampicillin/Sulbactam; Amp: Ampicillin; Cef: Cefazolin; No: no resistance or intermediate resistance. c Primer: vtx1: KS7/KS8, vtx2: GK5/GK6, eae: SK1/SK2, EHEC eae: LP1/LP3, EHEC hly A: hlyA1/hlyA4. d Intermediate resistant. ND: not determined. b

(1.4% agarose). After ethidium bromide staining they were visualized under UV light. A set of 13 antibiotics was selected for susceptibility testing following the recommendations of the National Committee for Clinical Laboratory Standards, USA (NCCLS, 2001) and according to the substances used in CDC studies (CDC, 1999). Testing was performed according to the NCCLS (2001) by using a broth microdilution method (NCCLS, 2000) in ready-to-use microtiter-plates with lyophilized antibiotics (MD Plate MG, MCS Diagnostics, Swalmen, The Netherlands). In short a 0.5 McFarland unit suspension in MuellerHinton-broth (Merck no. 110293, Darmstadt, Germany) was diluted 1:30 and an aliquot of 50 ml was inoculated into each well, which resulted in a final inoculum of about 5  105 cfu ml
1. The results were read after incubation for 16–20 h at 3770.51C. Each E. coli strain was tested with the following 13 substances (range in mg l
1): ampicillin [0.12–8], ampicillin/sulbactam (2:1) [8/4–16/8], cefazolin [2–16], cephalothin [2–16], ciprofloxacin [0.06–2], lomefloxacin [0.5–4], norfloxacin [4–8], ofloxacin [0.5–4], gentamicin [0.25–8], chloramphenicol [4–16], trimethoprim/sulfamethoxazole (1:19) [0.5/9.5–2/38], nitrofurantoin [32–64] and tetracycline [0.25–8]. E. coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were used as reference strains.

3. Results and discussion Fourty-one isolates out of the 60 strains were serotyped (the other 19 isolates were not available for serotyping). In total 25 different types could be identified. Most frequent was serotype O156:H47 (n ¼ 7; isolated from minced meat), followed by O1:H2 (n ¼ 3; isolated from minced meat) and O26:H11 (n ¼ 3; isolated from faeces (cattle) and sausage). The other 27 strains represented another 22 different serotypes, amongst them one O157:H7 strain.

Table 3 In vitro activity of antimicrobial agents against 60 E. coli isolates with VTEC associated virulence factors Antimicrobial agent

MIC range (mg ml
1)

MIC50 (mg ml
1)

MIC90 (mg ml
1)

Ampicillin Ampicillin/Sulbactam Cefazolin Cephalothin Ciprofloxacin

1–>8 o8/4–16/8 o2–>16 4–32 o0.06– 0.12 o0.5 o4 o0.5–1 o0.25–1 4–16 o0.5/9.5– 1/19 o32–64 2–>8

4 8/4 o2 16 o0.06

8 8/4 o2 32 o0.06

o0.5 o4 o0.5 o0.25 8 o0.5/9.5

o0.5 o4 o0.5 0.5 8 o0.5/9.5

o32 4

o32 8

Lomefloxacin Norfloxacin Ofloxacin Gentamicin Chloramphenicol Trimethoprim/ Sulfamethoxazol Nitrofurantoin Tetracycline

An overview concerning the relation between VTECAVF and origin of the strains is given in Tables 1 and 2. The results of the MIC tests indicated no relationship between serotype, virulence factor and antibiotic resistance (Table 2). Resistance only against cephalothin was most commonly observed (55%) and 12% of the isolates showed no resistance at all. Isolates from sausage showed the highest resistance. All isolates were susceptible to all fluoroquinolones tested, gentamicin, trimethoprim/sulfamethoxazole, nitrofurantoin and only one was resistant to cefazolin. Resistance could be detected for ampicillin (5% resistant or intermediate resistant), cephalothin (78%), tetracycline (27%) and chloramphenicol (8%, only intermediate resistant). The MIC (range and MICs for 50% [MIC50] and for 90% [MIC90] of isolates tested) of the antimicrobial agents evaluated are presented in Table 3. MICs at which 90% of the strains were inhibited were 8 mg ml
1 for ampicillin, 8/4 mg ml
1 for ampicillin/ sulbactam, >16 mg ml
1 for cephalothin, 8 mg ml
1 for tetracycline and 8 mg ml
1 for chloramphenicol.

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33

The resistance patterns of the tested strains with VTEC-associated virulence factors were comparable to those published for E. coli O157:H7 strains by the annual CDC studies in the United States for 1997–1999 (CDC, 1997, 1998, 1999) (Table 4). When selected antibiotic substances and their resistance profile are compared in detail the results of this investigation are in full agreement with the CDC studies (CDC, 1997, 1998, 1999). For substances used as therapeutics in human medicine (fluoroquinolones, aminoglycosides and trimethoprim/sulfamethoxazole) all four studies showed high susceptibility rates. On the other hand, data for E. coli isolates from various food and veterinary sources (clinical/nonclinical, animal husbandry, food; all isolates without VTEC-AVF) showed high resistance rates for those substances (reviewed by Doyle et al., 1997). For fluoroquinolones resistance rates were reported from 0% to 10%, for gentamicin from 70% to 90% and for cotrimoxazol at o5%. However, the relative low number of investigated isolates (this study n ¼ 60; CDC, 1997 n ¼ 161; CDC, 1998 n ¼ 313; CDC, 1999 n ¼ 292) limits the validity of this comparison as well as differences in the method. The origin of the majority of the strains in this study was cattle. The reason for this distribution was that isolates with VTEC-AVF are more often isolated from cattle than from other sources. Generally resistance rates in porcine and avian isolates are higher than in bovine isolates, as the therapeutic use of antibiotics is higher in these animal species (Trolldenier, 1996), but resistance against florfenicol was also reported from isolates from cattle originating from France and Germany (Cloeckaert et al., 2000) or from Spain (Orden et al., 2001). Another study (Altieri and Massa, 1999) also stated higher resistance rates in isolates from cattle than expected (e.g. tetracycline resistance up to 71.5%). Compared to those reports, resistance rates in this investigation with VTEC-AVF are low. The following substances did not show uniform MICs: ampicillin, cephalothin, chloramphenicol and tetracycline. Table 4 gives an overview on the percentage of resistant, intermediate and susceptible isolates for these substances compared to the CDC studies (CDC, 1997, 1998, 1999). For the substances tetracycline and especially cephalothin a high number of strains (11 out of 60 for tetracycline and 40 out of 60 for cephalothin) in this investigation were intermediate resistant, whereas the numbers of the CDC studies (Table 4) were considerably lower. However, the MICs were distributed in a narrow range. A slight shift in the MICs could result in a significant higher rate of resistant strains in the future. Indeed recently published data from CDC (2000) (n ¼ 407) indicate a tendency for higher resistance rates for chloramphenicol (4%) and tetracycline (7%) than in the years before.

31

Table 4 Rates of resistant, intermediate and susceptible E. coli isolates with VTEC-associated virulence factors in different studies Antimicrobial agent

Resistant isolates (%)

Intermediate isolates (%)

Susceptible isolates (%)

Ampicillin This studya CDC ‘97c CDC ‘98d CDC ‘99e

5b 0 2.6 1.4

0 0 0 0

95 100 97.4 98.6

Chloramphenicol This study CDC ‘97 CDC ‘98 CDC ‘99

0 0 0.3 0

8 0 0.3 0

92 100 99.4 100

Tetracycline This study CDC ‘97 CDC ‘98 CDC ‘99

8 3.1 4.5 3.4

Cephalothin This study CDC ‘97 CDC ‘98 CDC ‘99

10 3.7 0 0.6

18 0 0 0

73 96.9 95.5 96.6

67 6.2 4.8 5.8

23 90.1 95.2 94.6

a

n ¼ 60: The differentiation between intermediate and resistant was not possible because of the limited range of the antibiotic tested. c CDC (1997) n ¼ 161: d CDC (1998) n ¼ 313: e CDC (1999) n ¼ 292: b

Multi-drug-resistance (resistances against three or more antibiotics) in clinical isolates of E. coli can be found in rates of approx. 7% of the isolates (Sahm et al., 2001). Most of these multi-drug-resistant isolates (derived from human patients) were resistant to ampicillin, trimethoprim/sulfamethoxazol, cephalothin and ciprofloxacin. However, no multi-resistant isolates could be observed in this investigation. This observation is in agreement with reports from VTEC O157 isolates from humans in England and Wales, where only 2% of the isolates where multi-resistant (Threlfall, 2001). On the other hand, multiple resistance (in this case defined as resistance against two or more antibiotics) was found in 47% of VT positive isolates from pigs. Especially, tetracycline showed high resistance rates in that investigation (approx. 88%) (desRosiers et al., 2001). No common pattern of antimicrobial resistance was observed between human and porcine isolates in that study. The authors concluded, that the isolates from human or porcine origin were genetically not related. Another explanation may be, that antimicrobial therapy in pigs often includes tetracycline or other antibiotics resulting in higher resistance rates (Mathew et al., 1999)

32

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33

whereas human clinical infections with VTEC are normally not treated with antibiotics (Gill and Hamer, 2001). The resistance profile of E. coli with VTEC-AVF investigated in this study reveals a high percentage of susceptible isolates compared to E. coli isolates in general and is similar to profiles published for EHEC or VTEC strains in the literature. In general, the resistance to sulphonamides and tetracycline is increasing in incidence for E. coli (Threlfall, 2001). In the present investigations resistance to tetracyclines and cephalothin show the potential for increase, similar to the results of Sahm et al., (2001) for cephalothin and those of Threlfall (2001) for tetracycline. This may have only minor direct impact on human health, because antibiotic therapy in humans is unusual as mentioned above. However the potential increase of resistance within a population of generally susceptible isolates is undesirable with regard to the general public health policy to reduce antibiotic resistance in the food chain.

Acknowledgements We thank L. Br.autigam and H. Irsigler, Institut fur . Fleischhygiene und technologie, Freie Universit.at Berlin, for excellent technical assistance.

References Altieri, C., Massa, S., 1999. Antibiotic resistant E. coli from food of animal origin. Adv. Food Sci. 21, 166–169. Beutin, L., Bulte, . M., Weber, A., Zimmermann, S., Gleier, K., 2000. Investigation of human infections with verocytotoxin-producing strains of E. coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission. Epidemiol. Infect. 125, 47–51. . Bulte, . M., Heckotter, S., Schwenk, P., 1996. Enteroh.amorrhagische E. coli (EHEC)-aktuelle Lebensmittelinfektionserreger auch in der Bundesrepublik Deutschland? Nachweis von VTEC-St.ammen in Lebensmitteln tierischen Ursprungs. Fleischwirtschaft 76, 88–91. Caprioli, A., Busani, L., Martel, J.L., Helmuth, R., 2000. Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methods. Int. J. Antimicrob. Agents 14, 295–301. CDC, 1997. National antimicrobial resistance monitoring system: enteric bacteria. 1997 Annual Report, Centers for Disease Control, Atlanta, GA. CDC, 1998. National antimicrobial resistance monitoring system: enteric bacteria. 1998 Annual Report, Centers for Disease Control, Atlanta, GA. CDC, 1999. National antimicrobial resistance monitoring system: enteric bacteria. 1999 Annual Report, Centers for Disease Control, Atlanta, GA. CDC, 2000. National antimicrobial resistance monitoring system: enteric bacteria. 2000 Annual Report, Centers for Disease Control, Atlanta, GA. Cloeckaert, A., Baucheron, S., Flaujac, G., Schwarz, S., Kehrenberg, C., Martel, J.-C., Chaslus-Dancla, E., 2000. Plasmid-mediated florfenicol resistance encoded by the floR gene in E. coli isolated from cattle. Antimicrob. Agents Chemother. 44, 2858–2860.

desRosiers, A., Fairbrother, J.M., Johnson, R.P., Desautels, C., Letellier, A., Quessy, S., 2001. Phenotypic and genotypic characterization of E. coli verotoxin-producing isolates from humans and pigs. J. Food Prot. 64, 1904–1911. Doyle, M.P., Zhao, T., Meng, J., Zhao, S., 1997. E. coli O157:H7. In: Doyle, P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology—Fundamentals and Frontiers. ASM Press, Washington, DC, pp. 171–191. Farina, C., Goglio, A., Conedera, G., Minelli, F., Caprioli, A., 1996. Antimicrobial susceptibility of E. coli O157 and other enterohaemorrhagic E. coli isolated in Italy. Eur. J. Clin. Microbiol. Infect. Dis. 15, 351–353. Gill, C.J., Hamer, D.H., 2001. Foodborne illnesses. Curr. Treatment Opt. Gastroenterol. 4, 23–38. Hallmann, C., Wetzel, A., Reuter, G., 1997. Vorkommen von potentiell pathogenen E. coli in industriell hergestelltem Hackfleisch. In: Deutsche Veterin.armedizinische Gesellschaft e.V. (Ed.), Proceedings 38. Arbeitstagung Lebensmittelhygiene der DVG, Garmisch-Partenkirchen. Deutsche Veterin.armedizinische Gesellschaft, Gie
en, pp. 443–450. Holland, R.E., Wilson, R.A., Holland, M.S., Yuzbasiyan-Gurkan, V., Mullaney, T.P., White, D.G., 1999. Characterization of eae+ E. coli isolated from healthy and diarrheic calves. Vet. Microbiol. 66, 251–263. Kim, H.H., Samadpour, M., Grimm, L., Clausen, C.R., Besser, T.E., Bylor, M., Kobayashi, J.M., Neill, M.A., Schoenknecht, F.D., Tarr, P.I., 1994. Characteristics of antibiotic-resistant E. coli O157:H7 in Washington state, 1984–91. J. Infect. Dis. 170, 1606–1609. . Lehn, N., Stower-Hoffmann, J., Kott, T., Strassner, C., Wagner, H., . Kronke, M., Schneider-Brachert, W., 1996. Characterization of clinical isolates of E. coli showing high levels of fluoroquinolone resistance. J. Clin. Microbiol. 34, 597–602. Mathew, A.G., Saxton, A.M., Upchurch, W.G., Chattin, S.E., 1999. Multiple antibiotic resistance patterns of E. coli isolates from swine farms. Appl. Environ. Microbiol. 65, 2770–2772. Meng, J., Zhao, S., Doyle, M.P., Joseph, S.W., 1998. Antibiotic resistance of E. coli O157:H7 and O157:NM isolated from animals, food, and humans. J. Food Prot. 61, 1511–1514. Montenegro, M.A., Bulte, . M., Trumpf, T., Aleksi!c, S., Reuter, G., Bulling, E., Helmuth, R., 1990. Detection and characterization of fecal verotoxin-producing E. coli from healthy cattle. J. Clin. Microbiol. 28, 1417–1421. NCCLS, 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard— 5th Edition (M7-A5). NCCLS, Wayne, PA. NCCLS, 2001. Performance Standards for Antimicrobial Susceptibility Testing; 11th Informational Edition (M100-S11). NCCLS, Wayne, PA. OIE, 2001. Second International Conference on Antimicrobial Resistance. OIE, Paris, France. Paris, 2.-4.10.2001. Orden, J.A., Ruiz-Santa-Quiteria, J.A., Cid, D., D!ıez, R., Mart!ınez, S., de la Fuente, R., 2001. Quinolone resistance in potentially pathogenic and non-pathogenic E. coli strains isolated from healthy ruminants. J. Antimicrob. Chemother. 48, 421–424. Sahm, D.F., Thornsberry, C., Mayfield, D.C., Jones, M.E., Karlowsky, J.A., 2001. Multidrug-resistant urinary tract isolates of E. coli: prevalence and patient demographics in the United States in 2000. Antimicrob. Agents Chemother. 45, 1402–1406. Schmidt, H., Blaschke, B., Franke, S., Russmann, . H., Schwarzkopf, A., Heesemann, J., Karch, H., 1994. Differentiation in virulence patterns of E. coli possessing eae genes. Med. Microbiol. Immunol. 183, 23–31. Schmidt, H., Beutin, L., Karch, H., 1995. Molecular analysis of plasmid-encoded hemolysin of E. coli O157:H7 strain EDL 933. Infect. Immun. 63, 985–989.

G. Klein, M. Bulte . / Food Microbiology 20 (2003) 27–33 Stephan, R., Kuhn, . K., 1999. Prevalence of verotoxin-producing E. coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns. J. Vet. Med. B 46, 423–427. Stephan, R., Schumacher, S., 2001. Resistance patterns of non-O157 Shiga toxin-producing E. coli (STEC) strains isolated from animals, food and asymptomatic human carriers in Switzerland. Lett. Appl. Microbiol. 32, 114–117. Threlfall, E.J., 2001. Microbial drug resistance: implications for human health. Mitt. Geb. Lebensmittelunters. Hyg. 92, 59–67.

33

Threlfall, E.J., Ward, L.R., Frost, J.A., Willshaw, G.A., 2000. The emergence and spread of antibiotic resistance in food-borne bacteria. Int. J. Food Microbiol. 62, 1–5. Trolldenier, H., 1996. Resistenzentwicklungen von Infektionserregern landwirtschaftlicher Nutztiere in Deutschland (1990–1994)—ein . Uberblick. Dt. Tier.arztl. Wschr. 103, 256–260. Wong, C.S., Jelacic, S., Habeeb, R.L., Watkins, S.L., Tarr, P.I., 2000. The risk of the hemolytic-uremic syndrome after antibiotic treatment of E. coli O157:H7 infections. N. Engl. J. Med. 342, 1930–1936.