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JAC Advance Access originally published online on September 13, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1137-1141; doi:10.1093/jac/dkm316
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Characterization of CTX-M and SHV extended-spectrum ß-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia

Ahlem Jouini1,2, Laura Vinué2, Karim Ben Slama1, Yolanda Sáenz2, Naouel Klibi1, Salah Hammami3, Abdellatif Boudabous1 and Carmen Torres2,*

1 Laboratoire MBA, Département de Biologie, Faculté de Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia 2 Area de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain 3 Veterinary Research Institute, Tunis, Tunisia

* Corresponding author. Tel: +34-941299750; Fax: +34-941299721; E-mail: carmen.torres{at}unirioja.es

Received 4 May 2007; returned 26 June 2007; revised 25 July 2007; accepted 31 July 2007


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Objectives: To assess the occurrence of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli isolates of faecal samples of animals (n = 40) and food samples (n = 38) obtained in Tunisia in 2006, and to characterize the type of ESBLs, their genetic environments and the associated resistance genes.

Methods: Samples were inoculated in supplemented media (2 mg/L cefotaxime) for isolation of broad-spectrum cephalosporin-resistant E. coli isolates (one isolate/sample). ESBLs and their genetic environments as well as integrons and their gene cassette composition were characterized by PCR and sequencing.

Results: ESBL-producing E. coli isolates were detected in 10 of the 38 food samples analysed (26%) and in none of the tested animal faecal samples. Genes found were as follows (number of isolates): blaCTX-M-1 (5), blaCTX-M-1 + blaTEM-1b (1), blaCTX-M-14 + blaTEM-1b (2), blaCTX-M-8 (1) and blaSHV-5 (1). All ESBL-positive isolates showed unrelated PFGE patterns. ISEcp1 and IS903 were detected surrounding blaCTX-M-14, and ISEcp1/IS26 and orf477 surrounding some of the blaCTX-M-1 genes. Four of the ESBL-positive strains harboured class 1 integrons including different gene cassette combinations.

Conclusions: ESBLs, mainly of the CTX-M class, are detected in E. coli of food origin in Tunisia, being the first time that this mechanism has been detected in food E. coli strains in Africa.

Keywords: CTX-M-1 , CTX-M-8 , CTX-M-14 , SHV-5 , E. coli , animals


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The detection of clinical Escherichia coli isolates producing extended-spectrum ß-lactamases (ESBLs) has been increasingly reported in the last few years in a wide variety of countries, including Tunisia,1–3 and represents a clinical problem in human medicine.2 In addition, a new class of ESBL enzymes has emerged, the CTX-M class, which is dramatically spreading among human clinical E. coli isolates, mainly in those recovered from the community.1,4 A few reports have recently appeared referring to the detection of ESBL-containing E. coli strains in animal and food samples in different countries of Europe and Asia,5–8 but to our knowledge, never in Tunisia or in other African countries. The objective of our study was to detect ESBL-containing E. coli in faecal samples of food-producing animals as well as in food samples of animal origin in Tunisia and to characterize the type of ESBL enzymes, their genetic environments and the associated resistance genes.


   Materials and methods
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Samples and E. coli isolates

A total of 40 faecal samples of healthy animals were recovered either from farm animals (6 chickens, 16 cattle and 14 horses) or from veterinary clinics (2 cats and 2 dogs). In those samples obtained in farms, one lot of animals (50–60 animals for chickens, 15–20 for cattle and 1–2 for horses) was considered as one unique sample. In addition, 38 food samples of animal origin were also included in this study, and they were obtained either from different supermarkets or from a food laboratory of Tunis (23 samples of beef, 8 of chicken, 2 of turkey, 1 of sheep and 4 of fish). All samples of food and animal origin were obtained in the North of Tunisia during 2006.

Samples were suspended in sterile saline solution and then were seeded on MacConkey agar plates supplemented with 2 mg/L cefotaxime and incubated for 24 h at 37°C. Isolates with typical E. coli morphology were selected (one per sample) and identified by classical biochemical methods, as well as by a species-specific PCR of the uid gene encoding ß-glucuronidase (primers used: uid-F, 5'-ATCACCGTGGTGACGCATGTCGC-3'; and uid-R, 5'-CACCACGATGCCATGTTCATCTGC-3').

Antimicrobial susceptibility testing

E. coli isolates recovered from the antibiotic-supplemented MacConkey agar plates were submitted to susceptibility testing for 17 antimicrobial agents by the disc diffusion method according to the CLSI criteria.9 The antimicrobial agents tested were as follows (µg): ampicillin (10), amoxicillin/clavulanic acid (20 + 10), cefoxitin (30), ceftazidime (30), cefotaxime (30), aztreonam (30), imipenem (10), gentamicin (10), amikacin (30), tobramycin (10), streptomycin (10), nalidixic acid (30), ciprofloxacin (5), sulfamethoxazole (300), trimethoprim/sulfamethoxazole (1.25 + 23.75), tetracycline (30) and chloramphenicol (30). The MICs of nalidixic acid and ciprofloxacin were also determined by the agar dilution method in all quinolone-resistant E. coli isolates.9 A screening test for the detection of ESBLs was carried out by the double disc diffusion test (using cefotaxime, ceftazidime and amoxicillin/clavulanic acid discs) according to the CLSI criteria.9 E. coli ATCC 25922 was used as a quality control strain.

Characterization of ß-lactamase genes and genetic environment of blaCTX-M genes

The presence of genes encoding TEM (F, 5'-ATTCTTGAAGACGAAAGGGC-3'; R, 5'-ACGCTCAGTGGAACGAAAAC-3'; amplicon size 1150 bp), SHV (F, 5'-CACTCAAGGATGTATTGTG-3'; R, 5'-TTAGCGTTGCCAGTGCTCG-3'; amplicon size 885 bp), OXA-1 (F, 5'-ACACAATACATATCAACTTCGC-3'; R, 5'-AGTGTGTTTAGAATGGTGATC-3'; amplicon size 813 bp), CTX-M-1 group (F, 5'-GTTACAATGTGTGAGAAGCAG-3'; R, 5'-CCGTTTCCGCTATTACAAAC-3'; amplicon size 1041 bp), CTX-M-8 group (F, 5'-TGATGAGACATCGCGTTAAG-3'; R, 5'-TAACCGTCGGTGACGATTTT-3'; amplicon size 875 bp), CTX-M-9 group (F, 5'-GTGACAAAGAGAGTGCAACGG-3'; R, 5'-ATGATTCTCGCCGCTGAAGCC-3'; amplicon size 857 bp) and CTX-M-10 (F, 5'-CCGCGCTACACTTTGTGGC-3'; R, 5'-TTACAAACCGTTGGTGACG-3'; amplicon size 944 bp) ß-lactamases was analysed by PCR and sequencing (on both strands).5,6 Nucleotide segments and their deduced amino acid sequences were compared with those included in the GenBank database as well as with those deposited at the web site http://www.lahey.org/Studies/, in order to ascribe the specific type of ß-lactamase gene. Positive and negative controls were included in all PCR assays. The presence of ISEcp1, IS26, orf477 or IS903 sequences surrounding the blaCTX-M genes was analysed by PCR using primers and conditions as previously described;10 all obtained amplicons were sequenced for confirmation.

The promoter and attenuator region of the chromosomal ampC gene was also amplified by PCR, sequenced and compared with the same region of the E. coli K12 ampC gene, to analyse the mutations associated with the overexpression of this gene.5

Characterization of integrons and resistance mechanisms to non-ß-lactam antimicrobial agents

The presence of genes associated with tetracycline [tet(A) and tet(B)], streptomycin [aadA1 and aadA2], sulfamethoxazole [sul1, sul2 and sul3] and gentamicin [aac(3)-I, aac(3)-II and aac(3)-IV] resistance was studied by PCR.11 gyrA and parC genes were amplified by PCR in all quinolone-resistant E. coli isolates, and the obtained sequences were compared with those previously reported for gyrA (GenBank accession number X06373) and parC (M58408 with the modification included in L22025) genes.11 The presence of the genes encoding class 1 and class 2 integrases was examined by PCR and the gene cassettes included in the variable region of class 1 integrons were detected by PCR and sequencing.11

PFGE patterns of E. coli isolates

The clonal relationship between the strains was studied by PFGE, using XbaI as a restriction enzyme.11


   Results and discussion
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Seventy-eight food and animal faecal samples were analysed to detect the presence of ESBL-producing E. coli isolates using cefotaxime-supplemented agar plates. Broad-spectrum cephalosporin-resistant E. coli isolates were recovered from 11 of these samples (14%), and ESBL-containing E. coli isolates were detected in 10 of them (13%), when the double disc screening test was used (Table 1). All of them were isolated from samples of food origin, representing 26% of the total food samples analysed in this study. No colonization by ESBL-containing E. coli isolates was detected among the tested animal faecal samples. The difference observed in the prevalence of ESBL-positive E. coli isolates in faecal animal samples in relation to the food animal samples might have different reasons. The animals from which food and faecal samples were obtained were grown in non-related farms. On the other hand, although the E. coli contamination of food is expected to be due to faecal E. coli at the slaughterhouse level, contamination during the transformation or commercialization processes of food cannot be excluded.


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  		Table 1. Characteristics of the 11 broad-spectrum cephalosporin-resistant E. coli strains detected in 11 food samples of animal origin in Tunisia

 
Characterization of ESBL genes in E. coli isolates of food origin

The ß-lactamase genes detected by PCR and sequencing in the 10 ESBL-positive E. coli isolates were as follows: blaCTX-M-1 (five isolates, four of beef and one of turkey origin), blaCTX-M-1 + blaTEM-1b (one isolate of beef), blaCTX-M-14 + blaTEM-1b (two isolates of chicken), blaCTX-M-8 (one isolate of chicken) and blaSHV-5 (one isolate of chicken) (Table 1). All the ESBL-positive E. coli isolates showed unrelated PFGE patterns. It is of interest to underline that these detected CTX-M ß-lactamases belong to different groups (groups 1, 8 and 9), including the very unusual CTX-M-8 ß-lactamase. The genetic location of blaCTX-M genes has not been studied in this work, but other authors have demonstrated the plasmidic location of these genetic determinants.2

Genetic environments of blaCTX-M genes

Figure 1 shows the genetic environments of blaCTX-M genes detected among our ESBL-positive E. coli isolates. orf477 was detected downstream of blaCTX-M-1 in all six strains that harboured that ESBL gene. The sequence of the fragment obtained by PCR upstream of the blaCTX-M-1 gene in three of the E. coli strains (C920, C924 and C925) revealed the presence of a region of the IS26 transposase (211 bp) flanking a partially truncated ISEcp1 whose size was 214 bp and followed by an intergenic region (32 + 48 bp, named by Eckert et al.10 as X+W sequences, respectively). This whole structure was similar (98%) to that previously described in E. coli 5402 with the EMBL accession number AJ416342. The upstream region of the blaCTX-M-1 gene in the remaining three E. coli strains (C922, C923 and C926) could not be identified because all PCRs performed were negative. The presence of ISEcp1 and IS903 was demonstrated upstream and downstream, respectively, of the blaCTX-M-14 gene in E. coli C929 and C930, and an intergenic region of 42 bp (previously named as the Y region10) was found between ISEcp1 and blaCTX-M-14 (Figure 1).


Figure 1
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  		Figure 1. Genetic environments of blaCTX-M genes detected among our ESBL-positive E. coli strains (the intergenic X, Y and W regions have been previously reported10).

 
All PCRs for detection of ISEcp1, IS26, orf477 or IS903 sequences surrounding the blaCTX-M-8 gene were negative in our E. coli strain C921. The role of IS26 and ISEcp1 insertion sequences in the mobilization and insertion of blaCTX-M genes has been previously described.10

Mechanism of resistance in a broad-spectrum cephalosporin-resistant and ESBL-negative E. coli strain

A broad-spectrum cephalosporin-resistant ESBL-negative strain (E. coli C927) was recovered in a turkey food sample of this study. blaTEM-1b was the unique acquired ß-lactamase gene detected in this strain. The promoter/attenuator region of the chromosomal ampC gene of this strain was sequenced and three changes in the nucleotide sequence were detected at positions –28, +52 and +72, but no mutations were observed at the important –42 and –32 positions, associated with broad-spectrum cephalosporin resistance.5

Mechanism of resistance to non-ß-lactam antimicrobial agents

Table 1 shows the pattern of resistance to non-ß-lactam antimicrobial agents in all 11 broad-spectrum cephalosporin-resistant E. coli strains. Six of the strains showed resistance to at least three of the tested non-ß-lactam antimicrobial agents. A variety of resistance genes were observed among our strains: tet(A) or tet(B) (in the seven tetracycline-resistant strains), aadA1 with/without aadB (in five streptomycin-resistant strains), sul1 with/without sul2 (in five sulphonamide-resistant strains), aac(3)-II (in three gentamicin-resistant strains) and dfrA1 (in three trimethoprim/sulfamethoxazole-resistant strains). The presence of class 1 integrons was demonstrated in 5 of the 11 cephalosporin-resistant strains and two different gene cassette combinations were identified (Table 1).

Amino acid changes in GyrA and ParC proteins were observed among our six quinolone-resistant strains. Five of the strains showed nalidixic acid resistance (MICs of 128–512 mg/L), being ciprofloxacin susceptible (MICs of 0.125–0.5 mg/L), and one amino acid change in the GyrA protein (but not in ParC) was observed (Ser-83 -> Leu or Ser-83 -> Ala or Asp-87 -> Tyr). The remaining quinolone-resistant strain (E. coli C930) showed a high-level nalidixic acid and ciprofloxacin resistance phenotype (MICs of ≥ 2048 and 128 mg/L, respectively), and two amino acid changes in both GyrA (Ser-83 -> Leu + Asp-87 -> Asn) and ParC (Ser-80 -> Ile + Glu-84 -> Gly) proteins were identified (Table 1).

Conclusions

A high percentage of food samples were colonized by ESBL-containing E. coli strains (26%), mainly of the CTX-M class. In addition, certain associations between animal species and CTX-M types seem to occur: CTX-M-1 has been detected in strains recovered from food samples of beef origin and the CTX-M-14 or CTX-M-8 enzymes in those of chicken origin. The use of cephalosporins in food-producing animals could be a selective factor for the appearance of ESBL-containing bacteria in animals. Nevertheless, due to the fact that most of the ESBL-containing E. coli isolates recovered either from animals or from humans harbour a wide variety of resistance genes to non-ß-lactam antimicrobial agents, the possibility of co-selection by the use of non-ß-lactams could happen and this fact should be extensively evaluated in the future.

To our knowledge, this is the first report describing ESBL-producing E. coli strains detected in samples of food origin in Tunisia and in the African continent. In addition, this is the first time that the unusual CTX-M-8 ß-lactamase has been detected in bacteria of non-human origin. It seems that CTX-M ß-lactamases are spreading not only among animals and humans in Europe and Asia, but also in Tunisia. More studies should be carried out in the future to track the evolution of these types of ß-lactamases in isolates of different origins in different countries of the African continent.


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This study was funded by the Spanish Agency of International Collaboration (AECI, Projects A/3989/05 and A/5300/06) from the Ministerio de Asuntos Exteriores of Spain and from the Tunisian Ministry of Higher Education, Scientific Research and Technology. This work was also financed by the project SAF2006-14207-C02 from the Ministry of Science and Education of Spain.


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None to declare.


   References
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1 Livermore DM, Cantón R, Gniadkowski M, et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother (2007) 59:165–74.[Abstract/Free Full Text]

2 Paterson DL, Bonomo RA. Extended-spectrum ß-lactamases: a clinical update. Clin Microbiol Rev (2005) 18:657–86.[Abstract/Free Full Text]

3 Mamlouk K, Boutiba-Ben B, Gautier V, et al. Emergence and outbreaks of CTX-M ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol (2006) 44:4049–56.[Abstract/Free Full Text]

4 Cantón R, Coque TM. The CTX-M ß-lactamase pandemic. Curr Opin Microbiol (2006) 9:466–75.[CrossRef][Web of Science][Medline]

5 Briñas L, Moreno MA, Teshager T, et al. Monitoring and characterization of extended-spectrum ß-lactamases in Escherichia coli strains from healthy and sick animals in Spain in 2003. Antimicrob Agents Chemother (2005) 49:1262–4.[Abstract/Free Full Text]

6 Costa D, Poeta P, Sáenz Y, et al. Detection of Escherichia coli harbouring extended-spectrum ß-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J Antimicrob Chemother (2006) 59:1311–2.

7 Kojima A, Ishii Y, Ishihara K, et al. Extended-spectrum-ß-lactamase-producing Escherichia coli strains isolated from farm animals from 1999 to 2002: report from the Japanese Veterinary Antimicrobial Resistance Monitoring Program. Antimicrob Agents Chemother (2005) 49:3533–7.[Abstract/Free Full Text]

8 Meunier D, Jouy E, Lazizzera C, et al. CTX-M-1- and CTX-M-15-type ß-lactamases in clinical Escherichia coli isolates recovered from food-producing animals in France. Int J Antimicrob Agents (2006) 28:402–7.[CrossRef][Web of Science][Medline]

9 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Seventeenth Informational Supplement M100-S17 (2007) Wayne, PA, USA: CLSI.

10 Eckert C, Gautier V, Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother (2006) 57:14–23.[Abstract/Free Full Text]

11 Sáenz Y, Briñas L, Domínguez E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal and food origins. Antimicrob Agents Chemother (2004) 48:3996–4001.[Abstract/Free Full Text]


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