European Food Research and Technology

, Volume 226, Issue 3, pp 371–375

Antimicrobial resistance in Escherichia coli strains isolated from organic and conventional pork meat: a comparative survey

Authors

  • J. M. Miranda
    • Laboratorio de Higiene Inspección y Control de Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria pabellónUniversidad de Santiago de Compostela
  • B. I. Vázquez
    • Laboratorio de Higiene Inspección y Control de Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria pabellónUniversidad de Santiago de Compostela
  • C. A. Fente
    • Laboratorio de Higiene Inspección y Control de Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria pabellónUniversidad de Santiago de Compostela
  • J. Barros-Velázquez
    • Area de Tecnología de los Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de VeterinariaUniversidad de Santiago de Compostela
  • A. Cepeda
    • Laboratorio de Higiene Inspección y Control de Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria pabellónUniversidad de Santiago de Compostela
    • Laboratorio de Higiene Inspección y Control de Alimentos, Dpto de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria pabellónUniversidad de Santiago de Compostela
Original Paper

DOI: 10.1007/s00217-006-0547-y

Cite this article as:
Miranda, J.M., Vázquez, B.I., Fente, C.A. et al. Eur Food Res Technol (2008) 226: 371. doi:10.1007/s00217-006-0547-y

Abstract

Mean counts of Escherichia coli were determined for 54 samples of organic pork meat, and in 67 samples of conventional pork meat. Up to three E. coli strains from each sample were analysed by an agar disk diffusion assay for their resistance to ampicillin, chloramphenicol, cephalotin, doxycycline, enrofloxacin, gentamicin, nitrofurantoin, sulfisoxazole and streptomycin by the agar disk diffusion method. Results indicated that the presence of E. coli was significantly (< 0.05) higher in organic pork meat as compared to conventional pork meat. Isolates from organic pork meat exhibited lower levels of antimicrobial resistance against ampicillin (< 0.0001), doxycycline (< 0.0001) and sulfisoxazole (< 0.0001), as compared to isolates from conventional meat. Moreover, presence of multi-resistant E. coli strains was significantly (< 0.0001) higher in conventional pork meat as compared to organic pork meat, the largest differences being observed for isolates resistant to combinations of ampicillin, sulfisoxazol and/or doxycycline. Organically-farmed pork samples showed significantly lower development of antimicrobial resistance in E. coli, thus contributing to reduce the development and spread of antimicrobial resistance among these food-borne bacteria.

Keywords

E. coliOrganicAntimicrobialResistancePorkMeat

Introduction

Organic and other non-conventional meat products are now readily available for retail in developed countries, to satisfy consumers’ demand of high-quality products that meet the following requirements: (1) guaranteeing animal welfare during production; (2) absence of chemical agents during animal feeding; (3) environmental consideration and (4) better taste than conventional products [7]. In addition, there is a perception on a part of consumers that due to the fact that conditions for growth are more natural for organic farming than for the other one meat produts will have less pathogenic bacteria [5]. However, little is known about microbiological status of organic animal products and the potential microbiological risks linked to organic meat production. Thus, raising of animals outdoors, use of slow-growing breeds, the strict restrictions in therapeutic use of antimicrobial agents and use of very small slaughtering facilities may not guarantee a strict microbiological control of animals destined to human consumption [7, 19].

It has been previously reported that several antimicrobial-resistant bacteria isolated from humans primarily originated from animals raised for human consumption [1, 4] and that such resistant bacteria may contaminate meat derived from those animals [15]. Although this populations of resistant bacteria declines in the absence of the antimicrobial agent [14], antimicrobial-resistant bacteria may persist in meat even after the withdrawal period [24, 28]. Thus, antibiotic resistance of bacterial isolates from animal origin can also represent a potential hazard to consumers via food-borne infections caused by antibiotic-resistant bacteria.

Monitoring of the use of antimicrobial agents in veterinary medicine in animals destined for human consumption is considered to be a risk-management option to prevent development and spread of antimicrobial resistance in microorganisms present in food-producing animals [26]. In this sense, Escherichia coli has been described to be a very useful biomarker to evaluate the development of antimicrobial resistance [25]. This is due to the high frequency of mutation exhibited by E. coli leading to the development of antimicrobial resistance, as compared to other microorganisms frequently found in animals and foods [10, 15, 25].

Recently, other authors have reported of antimicrobial resistance by bacteria isolated from organic animal products. The data reported in such studies are referred to poultry and dairy products [5, 6, 16, 17, 19, 21]. However, little information relative to organic pork meat products is currently available. As a consequence of this, the main goal of this study was to investigate the prevalence of antimicrobial susceptibility of E. coli strains derived from organic pork as compared to conventional pork. The implications of these results in terms of microbiological safety, especially concerning the development and spread of antimicrobial resistance to the food chain, are also discussed.

Materials and methods

Collection of pork meat samples

A total of 121 loin boneless samples were taken during 2005 from supermarkets and butcher shops: 54 fresh pre-packaged organic-reared pork samples, and 67 fresh pre-packaged conventionally-reared pork samples. For the case of conventional pork, 4–5 samples of 14 commercial brands were used. All samples were taken from different lots in different supermarkets and butcher shops. For the case of organic pork, only three commercial brands certified as organic by a official agency were found. Thus, 18 samples were taken from each brand, all of them in different days. Samples were taken to the laboratory in an ice chest in less than an hour for immediate processing. All supermarkets and butcher shops were located in Galicia (north-western Spain).

Microbiological analyses

A total of 25 g portions was obtained from each pork meat sample, placed in a sterile masticator bag together with an appropriate volume (1/9) (w/v) of sterile 0.1% peptone water (Merck, Darmstadt, Germany), and homogenized in a masticator (AES, Combourg, France) for 1 min. After homogenization, samples were cultured for the presence of E. coli. About 1 ml of 10−1 to 10−4 dilutions of meat extracts were processed on plates of Fluorocult® Agar prepared following the manufacturers’ instructions (Merck). Once the agar had solidified, the plates were overlaid with 3–4 ml of melted Fluorocult® and incubated at 44°C for 24 h. After incubation, pink to red colonies exhibiting blue florescence after exposure to a 365 nm ultraviolet lamp (Vilbert Lourmat, Marne, France) were identified as E. coli and counted.

Once bacterial counts were determined, one-to-three typical E. coli colonies isolated from each pork sample were picked, transferred onto Columbia agar supplemented with 5% sheep blood (BioMérieux, Marcy l’Etoile, France) and incubated at 44°C for 24 h in order to obtain pure cultures. Such pure cultures were identified by colony and cell morphology, Gram stain, methyl red stain, oxidase and catalase activity and indole production. Positive strains preliminary identified as E. coli were confirmed by the API 20E miniaturized identification tests (BioMérieux). All 180 E. coli isolates (90 derived from organic pork meat and 90 from conventional pork meat) were stored at −80°C in maintenance freeze medium units (Oxoid, Basingstoke, UK) until antimicrobial susceptibility was tested.

All meat samples were processed in triplicate. Sampling and processing of pork meat samples were always carried out by the same laboratory personnel. Agar media plates were prepared by the same research assistant throughout the study.

Antimicrobial susceptibility testing of bacteria

Antimicrobial susceptibility testing was performed for a total of 180 isolates of E. coli. Antimicrobial susceptibility testing was carried out by agar disk diffusion on Müeller–Hinton agar plates (Oxoid) according to Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guidelines [13]. Antimicrobial disks considered were ampicillin (10 μg), cephalotin (30 μg), chloramphenicol (30 μg), doxycycline (30 μg), enrofloxacin (5 μg), gentamicin (10 μg), nitrofurantoin (300 μg), streptomycin (10 μg) and sulfisoxazole (300 μg) (Oxoid). Antibiotic resistance breakpoints considered were those recommended by CLSI for veterinary pathogens [12], except for the case of nitrofurantoin and streptomycin, in which the CLSI interpretative criteria for Enterobacteriaceae were followed [13]. E. coli ATCC 25922 was used as a reference strain for this study.

Antimicrobial agents were selected in terms of their different structure and mechanism of action. E. coli isolates were classified as sensitive, intermediate (moderately sensitive) or resistant according to the criteria of the inhibition diameter zones established by the CLSI. Isolates exhibiting resistance to at least two of the antimicrobial agents tested were considered to be multi-resistant strains.

Statistical analysis

E. coli counts were grouped into three categories (<1 log CFU g−1; 1–2 log CFU g−1 and >2 log CFU g−1) prior to the performance of statistical analyses. The distributions of E. coli counts (log CFU g−1), as well as distributions of resistant strains were compared by means of the X2 test and Fisher’s exact test. The differences were considered to be significant when P was lower than 0.05. All statistical analyses were performed by means of the Statgraphics version 5.0.1. software (SAS Institute, North Carolina, USA).

Results

The results indicated that the special characteristics of organic production provided a higher prevalence of E. coli than conventional farming. Thus, for organic pork meat, E. coli was detected (>1 log CFU g−1) in 35 of the 54 samples tested (64.8% of the total). The counts determined were >2 log CFU g−1 for 9 of the 54 samples tested (16.7%), while 26 samples (48.1%) harboured E. coli in the 1–2 log CFU g−1 range. In the case of pork reared conventionally, E. coli was detected in only 33 of the 67 samples investigated (47.8% of the total). The counts determined in such samples were >2 log CFU g−1 in 2 of the 67 samples tested (3%), while 32 samples (47.8%) reached counts in the 1–2 log CFU g−1 range. According to these results, the E. coli counts obtained for organic pork samples were significantly higher (= 0.0231) than those obtained for conventional pork.

With respect to antimicrobial resistance of E. coli isolates, different patterns were observed in the strains isolated from organic pork samples as compared to their counterparts isolated from conventional pork (Table 1). Antimicrobial resistance was significantly higher in E. coli isolates derived from conventional pork samples for the antibiotics ampicillin (< 0.0001), doxycycline (< 0.0001), and sulfisoxazole (< 0.0001), as compared to E. coli counterparts isolated from organic pork (Table 1). In addition, althought significant differences between batches were observed with respect to cephalotin-resistance (= 0.0046), the results obtained for this antimicrobial agent were ambiguous. Thus, although a higher percentage of cephalotin-resistant microorganisms were isolated from conventional pork meat as compared to organic pork meat (14.4 vs. 6.7%), the latter products exhibited a lower presence of cephalotin-sensitive bacteria than the former (47.8 vs. 61.1%).
Table 1

Percentages of Escherichia coli strains isolated from conventional and organic pork that exhibited a sensitive (S), intermediate (I) or resistant (R) phenotype with respect to antimicrobial agents, as determined by agar disk diffusion method

Antimicrobial agent (μg)

Conventional pork meat (n = 90)

Organic pork meat (n = 90)

P

S

I

R

S

I

R

Ampicillin (10)

10

8.9

81.1

60

16.7

23.3

<0.0001

Cephalotin (30)

61.1

24.4

14.4

47.8

45.6

6.7

0.0046

Chloranphenicol (30)

94.4

3.3

2.2

93.3

1.1

5.6

0.2231

Doxycycline (30)

11.1

18.9

70

30

38.9

31.1

<0.0001

Enrofloxacin (5)

96.7

3.3

0

91.1

8.9

0

0.1366

Gentamicin (10)

91.1

8.9

0

91.1

7.8

1.1

0.5890

Nitrofurantoin (300)

93.3

5.6

1.1

91.1

6.7

2.2

0.8057

Sulfisoxazole (300)

28.9

1.1

70

72.2

7.8

20

<0.0001

Streptomycin (10)

4.4

68.9

26.7

3.3

53.3

43.3

0.0525

Similarly, organic pork meat showed a lower isolation rate of multi-resistant E. coli strains than conventional pork samples (Table 2). The percentage of E. coli isolates that exhibited resistance to at least two antimicrobial agents was lower (< 0.0001) in organic pork (41.1%) than in strains isolated from conventional pork (90%).
Table 2

Resistance patterns in Escherichia coli strains isolated from conventional and organic pork

Number of antimicrobials*

Conventional pork (n = 90)

No. (%) of isolates

Organic pork (n = 90)

No. (%) of isolates

0

2 (2.2)

26 (28.9)

1

7 (7.8)

27 (30)

2

28 (31.1)

21 (23.3)

3

39 (43.3)

12 (13.3)

4

11 (12.2)

2 (2.2)

≥5

3 (3.3)

2 (2.2)

Multi-resistant strains (%)

81 (90)

37 (41.1)

*The percentage of multi-resistant strains is expressed between brackets. Differences between conventional and organic pork were considered significant (< 0.0001)

Discussion

Results obtained in the present work indicated a relationship between the levels of antimicrobial resistance in E. coli and the tolerance of antimicrobial agents use in the pork production systems considered in this study. Thus, as expected for conventional pork, in which rearing procedures require more intensive antimicrobial consumption than organic pork farming, the average counts of E. coli were significantly lower than in the organic pork samples, in which the use of antimicrobial agents is seriously restricted. Recently, in function of the country of origin, widely variable rates of antimicrobial resistance were reported in E. coli isolated from pigs and pork meat conventionally farmed [3, 8, 9, 15, 18, 20]. The antimicrobial resistance rates determined in our study referred to conventionally-farmed pork are compatible with the results reported by these authors.

Other authors have described a correlation between percentages of antimicrobial resistant isolates and the use of antimicrobial agents in pork farming [1, 3]. The results obtained in our study provide evidence supporting the idea that prevalence and antimicrobial resistance of E. coli isolates differ depending on the type of animal production system (conventionally raised vs. organically raised) considered. Thus, while organic pork meat resulted to be more contaminated with E. coli, these isolates were more sensitive to certain antimicrobial agents than the counterpart E. coli strains isolated from conventionally-farmed animals. In this sense, the differences for the antimicrobial agents more commonly used in pork medicine, such as β-lactams (ampicillin), tetracyclines (doxycycline) and sulphonamides (sulfisozaxol) [3, 9, 20, 22, 23] should be noted. Moreover, the fact that no significant differences were observed for the antimicrobial agents banned for pork medicine in the EC such as chloranphenicol and nitrofurantoin provides additional support to the hypothesis that the development of antimicrobial resistance may be a direct consequence of clinical usage of certain antimicrobial agents.

Cross-resistance between β-lactams and tetracyclines or sulphonamides has been previously described [11, 25]. In the present work, the most frequent multi-resistance patterns found corresponded to combinations of ampicillin, sulfisozaxol and/or doxycycline.

Organic meat can demand higher prices in the market than conventional meat, and the sales of organic foods has increased in the recent years [27]. For this reason, the regulations concerning organic food production should ensure that meat derived from organic farming present in the markets are truly originated from organically reared animals. In this sense, the investigation of antimicrobial resistance could be a useful tool to detect fraudulent practices, since the detection of bacterial resistance to antimicrobial agents such as ampicillin, sulfisoxazol or doxycycline might point to the fraudulent substitution of organic pork meat with meat derived from conventional farming.

In summary, this work investigated the presence of antimicrobial resistance in microbial isolates obtained from pork meat derived from organic farming and conventional practices. The lower percentage of antimicrobial resistant E. coli isolates in organically raised pork supports organic pork rearing as a method to limit the presence of antibiotic resistance bacteria in food animals.

Acknowledgements

The authors wish to thank the Xunta de Galicia for granting two research projects (PGIDIT05TAL017E PGIDIT05TAL019E). The authors also thank Carmen Carreira for her expert technical assistance.

Copyright information

© Springer-Verlag 2006