Antibiotic resistance of Escherichia coli O157:H7 isolated from cattle and sheep

Abstract

A total of 102 Escherichia coli O157:H7 colonies recovered from 11 cattle and 14 sheep were collected and tested for their antibiotic resistance profiles using a disc diffusion method, according to the Clinical and Laboratory Standards Institute. Four (36.36 %) of the 11 cattle E. coli O157:H7 isolates were resistant to cephalothin, one (9.09 %) isolate was resistant to streptomycin, and one (9.09 %) to nalidixic acid. Two (14.28 %) of the 14 sheep E. coli O157:H7 isolates were resistant to sulphamethoxazole, one (7.14 %) isolate was resistant to sulphonamide compounds, and one (7.14 %) to streptomycin. All cattle and sheep isolates were found to be susceptible to cephazolin, gentamicin, ciprofloxacin, imipenem, trimethoprim/ sulphamethoxazole, chloramphenicol, trimethoprim, and ceftiofur. Six cattle isolates were susceptible at a ratio of 54.54 %, and 11 (78.57 %) isolates from sheep were susceptible to all 20 antibiotics tested. As an overall result, 68 % of the E. coli O157:H7 isolates belonging to cattle and sheep were susceptible to all antibiotics tested. On the other hand, most of the E. coli O157:H7 isolates were intermediately resistant to streptomycin, cephalothin, sulphamethoxazole, ampicillin, and kanamycin.

Introduction

Escherichia coli O157:H7 is considered one of the most important food-borne pathogens among shiga toxin-producing E. coli (STEC) strains. It causes diarrhea that may result in life-threatening conditions ranging from hemorrhagic colitis (HC) to hemolytic-uremic syndrome (HUS) (Mead et al. 1999; Meng et al. 2001). Gastrointestinal tracts of ruminants especially cattle and sheep have been shown to act as a reservoir of E. coli O157:H7 (Kudva et al. 1996, 1997; Shere et al. 1998). Epidemiological investigations have clearly associated E. coli O157:H7 human infections to the consumption of contaminated raw or undercooked ground beef and products with feces during slaughterhouse processing (Beutin et al.1993; Paiba et al. 2002).

Antimicrobial resistance of food-borne bacteria should not necessarily be considered distinct from that in isolates from humans, food animals, or other niches. When food animals, as carriers of asymptomatic E. coli O157:H7, are exposed to antimicrobial agents, they may become the reservoir of this antimicrobial-resistant bacteria. So it becomes important to determine whether the bacteria develop resistance to antimicrobials during food animal production. Also, recent studies have reported that there has been a rise in the antimicrobial resistance patterns of E. coli O157:H7 (Galland et al. 2001; Schroeder et al. 2002, 2004). It is controversial to use antibiotic treatment in humans to prevent HUS due to lysis of the bacteria and increased releasing of the expression of the shiga toxins in the intestinal tract (Takahashi et al. 1997; Wong et al. 2000). However, it is has been reported that using some antimicrobials in the early stage of infection may be protective against HUS progression (Fukushima et al. 1999; Ikeda et al. 1999).

The objective of this study was to evaluate the antibiotic resistance profile to 20 different antibiotics of E. coli O157:H7 isolates collected from cattle and sheep at slaughter for at least a 1-year period. As this is the first comprehensive study to evaluate the antibiotic resistance profile of E. coli O157:H7 in Turkey, it was possible to acquire epidemiological data for the future analysis.

Materials and methods

Bacterial strains

Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used for antibiotic susceptibility testing. E. coli O157:H7 ATCC 43895 was used as positive controls for the detection of fliC _h7 gene in PCR assay.

Isolates

A total of 102 E. coli O157:H7 isolates recovered from 25 animals including 11 cattle and 14 sheep were analyzed. The 41 isolates from the 11 cattle were obtained from 282 cattle (207 beef and 75 dairy cattle) feces and/or colon tissue samples between April 2002 and December 2003, and 61 isolates from 14 sheep were obtained from 218 sheep feces and/or colon tissue samples between November 2007 and November 2008 (with a total of 500 animals) (Table 1).

Table 1 Sampling design and antibiotic resistance profiles of E. coli O157:H7 isolates from cattle and sheep samples

IMS based cultural technique for the isolation of E. coli O157:H7

Immunomagnetic separation (IMS)-based selective enrichment technique was used for the isolation of E. coli O157 (Byrne et al. 2003). Briefly, 10 g of feces and/or colon tissue samples of cattle and sheep were weighed in a sterile bag and enriched with 90 ml EC broth (Oxoid CM0853, Hampshire, UK) containing novobiocin (20 µg/l; Sigma N-1628, St. Louis, MO, USA) and incubated at 37°C at 100 rpm for 18 h in a shaking incubator (Bellco Shel Lab Shaking Incubator S16R, Oregon, USA). After the incubation period, IMS was performed according to the manufacturer’s protocol. Then, 20 µl of magnetic beads coated with specific antibody against E. coli O157 (Dynabeads anti E. coli O157, Prod. No. 710.04; Dynal, Oslo, Norway) were used. Following the IMS procedure, 100 μl of PBS-Tween 20 was added to resuspend the bead and E. coli O157 complex.

Resuspended IMS mixture was plated on Sorbitol MacConkey Agar (Oxoid CM0813) plates containing Cefixime-tellurite supplement (Oxoid SR0172). After overnight incubation at 42°C, sorbitol negative colonies were tested for the O157 antigen by latex agglutination (Oxoid DR0620), and up to five agglutination positive colonies were taken and stored at −80°C (Sanyo MDF-U5186S, Japan) in cryovials for PCR analysis and antibiotic resistance determination.

DNA extraction and PCR analysis for detection of fliC _h7 gene

DNA extraction was performed using Chelex-100 (Bio-Rad, Hercules, CA, USA) resin-based technique as reported previously (Goncuoglu et al. 2010).

The PCR was performed with FLICH7-F and FLICH7-R primers in a final volume of 50 µl containing 1X Reaction Buffer (Promega, Madison, WI, USA), 1.5 mM MgCl₂ (Promega), 400 µM each of the four deoxynucleoside triphosphates (Promega), 2.5 U Taq DNA polymerase (Promega), 0.50 µM of primers (Promega) and 10 µl DNA. Thermal cycling (Biometra Personel Cycler, Göttingen, Germany) was carried out with the initial denaturation at 94°C for 2 min and then 35 cycles of denaturation at 94°C for 20 s, annealing at 54°C for 1 min, and extension at 72°C for 1 min, with a final extension for 10 min at 72°C (Fratamico et al. 2000). A 10-µl aliquot of each PCR product was subjected to 1.5 % agarose gel (SeaKem^® LE Agarose, Rockland, ME, USA) electrophoresis containing 0.1 µg/ml ethidium bromide for 1 h at 100 V. Amplicon visualization and documentation was performed using gel documentation and analysis system (Syngene Ingenius, Cambridge, UK).

Antibiotic susceptibility testing

The antibiotic susceptibility test of E. coli O157:H7 isolates was performed with the disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) (Anon 2006) in Mueller-Hinton agar (Oxoid CM0337) with 20 different antibiotics and antibiotic combinations at given concentrations (Oxoid) as shown in Table 2.

Table 2 Antibiotic resistance profiles of E. coli O157:H7 isolates from cattle and sheep feces and/or colon tissue samples

Escherichia coli O157:H7 isolates were transferred to BHI broth (Oxoid CM0225) and incubated at 37°C for 24 h. A loopfull of growth was transferred into 5 ml containing Tryptone Soya Broth (Oxoid CM0129). The culture was incubated at 37°C until it achieved the turbidity of the 0.5 McFarland standard. The correct density of the turbidity standard was measured by NanoDrop Spectrophotometer (NanoDrop ND-100, Delaware, USA). The absorbance of the broth cultures at 625 nm was fixed to 0.08–0.10. The suspension was inoculated uniformly to Mueller Hinton agar (uniform depth of 4 mm) with a sterile cotton swab. Then, antibiotic discs were placed onto the plate and incubated at 37°C for 18 h. According to the sizes of the inhibition zones, interpretation of the strains as susceptible, intermediate, or resistant was made according to the CLSI (Anon 2006).

Results and discussion

All the E. coli O157:H7 colonies (up to five colonies belonging to a positive sample) isolated from the same positive animals showed the same antibiotic resistance/susceptible profiles (Tables 1 and 2). Therefore, the results are given according to the number of the positive animals, e.g., 11 cattle and 14 sheep, instead of the total number (102) of the isolates tested in this study (Table 2). All isolates except one were recovered from healthy animals. Four of the 11 cattle isolates were recovered from cows. Nevertheless, no antibiotic resistance profile differences were seen between male and female host samples, one of the animals even showed enteritis.

Overall, among the cattle isolates, 36.36 % (4/11) of them were resistant to cephalothin, 9.09 % (1/11) were resistant to streptomycin and 9.09 % (1/11) were resistant to nalidixic acid. In one (9.09 %) E. coli O157:H7 cattle isolate, multiple drug resistance was shown to cephalothin and nalidixic acid. Also, ten isolates (90.90 %) were intermediately resistant to streptomycin, eight (72.72 %) were intermediately resistant to kanamycin, seven (63.63 %) were intermediately resistant to sulphamethoxazole, cephalothin, and ampicillin. In addition, five (45.45 %) isolates were intermediately resistant to amikacin, three (27.27 %) were intermediately resistant to sulphonamide compounds, and two different isolates (9.09 %) were intermediately resistant to amoxicillin/clavulanic acid and cefoxitin, respectively.

Among those isolates recovered from sheep, two (14.28 %) were resistant to sulphamethoxazole, one (7.14 %) to streptomycin, and one (7.14 %) to sulphonomide compounds. In one (7.14 %) E. coli O157:H7 sheep isolate, multiple drug resistance was shown to streptomycin and sulphonomide compounds. From total sheep isolates, eleven (78.57 %) of them were intermediately resistant to streptomycin, ten(71.42 %) were intermediately resistant to cephalothin, seven (50.00 %) were intermediately resistant to sulphamethoxazole, and five (35.71 %) were intermediately resistant to ampicillin. For all that, three (21.42 %) isolates were intermediately resistant to tetracycline and nalidixic acid, respectively. In addition, two (14.28 %) isolates were intermediately resistant to amikacin and kanamycin, respectively, and one (7.14 %) was intermediately resistant to ceftriaxone.

All isolates from cattle and sheep were susceptible to cephazolin, gentamicin, ciprofloxacin, imipenem, trimethoprim/sulphamethoxazole, chloramphenicol, trimethoprim, and ceftiofur. Among those isolates recovered from cattle, six (54.54 %) were susceptible to all 20 antibiotics tested. On the other hand, these susceptible isolates were all intermediately resistant to cephalothin and streptomycin. Regarding sheep isolates, eleven (78.57 %) isolates were susceptible to all tested antibiotics. Similar to the cattle isolates, most of the susceptible sheep isolates (ten, 71.42 %) were intermediately resistant to streptomycin.

Similar to the previous studies, in this study, susceptibility was amore common character instead of showing resistance to tested antibiotics (Meng et al. 1998; Zhao et al. 2001; Schroeder et al. 2002; Walsh et al. 2006). In fact, no multiresistant character was observed in all the isolates tested but, in agreement with Vali et al. (2004), a low prevalence of resistance to cephalothin, sulphamethoxazole, streptomycin, sulphonamide compounds, and nalidixic acid, was detected. Also, cephalothin and nalidixic acid resistance ratios were similar to Khan et al.'s (2002) findings. All isolates were susceptible to tetracycline used commonly on cattle and sheep feedlots as well as to chloramphenicol. Scott et al. (2006) showed that E. coli O157:H7 strains isolated from cattle feedlots were susceptible to 13 tested antibiotics. In particular, different studies (Kim et al. 1994; Galland et al. 2001; Schroeder et al. 2002; Wilkerson et al. 2004) have evidenced that only from 6.63 to 38.46% of E. coli O157:H7 isolates from bovines were resistant to tetracycline. Various countries including Turkey have banned the usage of chloramphenicol for food-producing animals, so this result is good data for chloramphenicol resistance. Sheep isolates were more susceptible to antibiotics than cattle isolates. This difference may be explained by the slaughtering age of the animals that reach up to 1 year old for sheep and more than 1 year old for cattle. Moreover, since intensive sheep breeding does not occur generally in Turkey, the antibiotics are not widely used on farms for animal prophylaxis, particularly for sheep.

Although ciprofloxacin, aminoglycosides and trimethoprim/sulphamethoxazole are used as therapeutics in human medicine, our isolates showed high susceptibility rates, except streptomycin and kanamycin with high intermediate resistant patterns. This variation in results may be due to attaining resistance from other organisms by genetic linkage of genetic factors, or by being from different origin, or these resistance patterns are the natural characteristic of the bacteria.

Although some of the isolates were recovered on the same slaughter day, the antibiotic resistance patterns of the isolates from different animals were quite distinct (Table 1). The antibiotic resistance profiles of four cattle isolates, which were coded as 1005 to 1021, were all different from each other. One of the isolates was resistant to streptomycin and another to cephalothin. On the other hand, two isolates showed no resistance to any antibiotic tested; also, all the four isolates were intermediately resistant to different antibiotics. Similarly, among the sheep isolates coded as 1073–1102, the antibiotic resistance profiles did not have the same pattern. Also, the intermediate resistance profiles of the isolates were all different from each other; in isolates 1078–1081, the only intermediate resistance was to streptomycin, but isolates 1100 to 1102 were intermediately resistant to nine different antibiotics.

According to the previous studies conducted worldwide on this subject, most isolates that are resistant to antibiotics are widely used on farms in food animals, more commonly used as prophylactics and therapeutics, but our results were unforeseeable. In the present study, most of the isolates showed an intermediate resistant profile which is a clue for potential resistance risk that may occur in the future. Therefore, attention has to be paid to high intermediate resistant profiles of these antibiotics.

This is the first report of antibiotic resistance patterns of E. coli O157:H7 strains isolated from cattle and sheep in Turkey. It is concluded that the overall prevalence of antibiotic resistance of E. coli O157:H7 isolates recovered from cattle and sheep tested in this study is very low. However, longitudinal studies should be performed to monitor and detect any changing in antibiotic resistance profiles of this bacterium in the future.