Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia

International Journal of Food Microbiology 203 (2015) 86–92

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia Leila Ben Said a, Ahlem Jouini b, Naouel Klibi a, Raoudha Dziri a, Carla Andrea Alonso c, Abdellatif Boudabous a, Karim Ben Slama a,d, Carmen Torres c,⁎ a

Laboratoire des Microorganismes et Biomolécules actives, Faculté de Sciences de Tunis, Université de Tunis El Manar, 2092 Tunis, Tunisia Laboratory of Epidemiology and Veterinary Microbiology, Pasteur Institute of Tunis, Tunisia c Area de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain d Institut Supérieur des Sciences Biologiques Appliquées de Tunis, Université de Tunis El Manar, 2092 Tunis, Tunisia b

a r t i c l e

i n f o

Article history: Received 11 October 2014 Received in revised form 22 January 2015 Accepted 20 February 2015 Available online 6 March 2015 Keywords: ESBL Enterobacteriaceae E. coli Soil Water Vegetables

a b s t r a c t One-hundred-nine samples of 18 different farms (49 of food-vegetables, 41 of soil and 19 of irrigation water) and 45 vegetable food samples of 13 markets were collected in Tunisia. These samples were inoculated in MacConkey agar plates supplemented with cefotaxime (2 μg/ml). ESBL-producing Enterobacteriaceae (ESBL-Eb) were detected in 10 of the 109 farm samples (vegetables, 8.2%; soil, 7.3%; water, 15.8%), and in 4 of 45 vegetables of markets (8.9%), recovering 15 ESBL-Eb. Isolates and ESBL genes detected were: Escherichia coli (n = 8: 5 blaCTX-M-1, 2 blaCTX-M-15 and one blaCTX-M-14), Citrobacter freundii (n = 4: 3 blaCTX-M-15 and one blaSHV-12), Enterobacter hormaechei (n = 2: 2 blaCTX-M-15) and Klebsiella pneumoniae (n = 1, blaCTX-M-15). The ISEcp1 sequence was found upstream of blaCTX-M genes in 13 of 14 strains (in three cases truncated by IS5), and orf477 or IS903 downstream. Class 1 integrons were detected in five strains and contained two gene cassette arrangements (dfrA17-aadA5 and aadA1). Most isolates tested showed a multiresistant phenotype. All blaCTX-M-15-positive strains carried the aac(6′)-1b-cr gene, that affects to amikacin–tobramycin–kanamycin–ciprofloxacin. Five ESBL-Eb strains carried genes of the qnr family. The 8 ESBL-positive E. coli isolates were typed as: ST58/B1 (n = 3) and ST117/D, ST131/B2, ST10/A, ST23/A, and the new ST3496/D (one strain, each). From 1–2 plasmids were detected in all ESBL-positive E. coli isolates (63–179 kb). The ESBL genes were transferred by conjugation in 4 blaCTX-M-1-positive E. coli strains, and transconjugants acquired a 97 kb IncI1 plasmid. ESBL-Eb isolates are frequently disseminated in vegetable farms and potentially could be transmitted to humans through the food chain. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Enterobacteriaceae form part of the normal microbiota of humans and animals, being Escherichia coli one of the most representative species of this family. These bacteria can spread through the fecal material and wastewater in different environments (Bain et al., 2014), including soil, vegetables, and others. Antibiotic resistance is an important problem of public health. Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-Eb) have emerged in the last decade as a global threat for human health (Pitout and Laupland, 2008). ESBL-Eb are not only isolated from hospital settings, but they are also disseminated in other environments as farm-animals and derived foods, domestic and even ⁎ Corresponding author at: Área de Bioquímica y Biología Molecular Universidad de La Rioja Madre de Dios, 51 26006 Logroño, Spain. Tel.: + 34 941299750; fax: + 34 941299721. E-mail address: [email protected] (C. Torres).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.02.023 0168-1605/© 2015 Elsevier B.V. All rights reserved.

in wild animals, healthy humans, wastewater, vegetables and other sources (Ben Sallem et al., 2012; Blaak et al., 2014; Jouini et al., 2007; Poeta et al., 2009; Vinué et al., 2009). The high use of antibiotics not only in human medicine, but also in veterinary medicine or even in agriculture, could constitute a selective pressure for the spread of antibiotic resistant bacteria, including ESBL-Eb (Durso and Cook, 2014). In the environment of the farms, vegetables could be contaminated with ESBL-Eb by the use of inadequately amended manure or by irrigation water, especially, if treated wastewater is used, which could constitute a reservoir of antibiotic resistant bacteria (Pignato et al., 2009; Selvaratnam and Kunberger, 2004). ESBL-Eb could persist in the surface of plants or even reach their interior, and they could be transmitted to humans or animals. It is very important to highlight the potential role of consumption of uncooked vegetables in gastrointestinal bacteria acquisition (Hamilton-Miller and Shah, 2001). There are few studies about the dissemination of ESBL-Eb in vegetables and in agricultural environments (Blaak et al., 2014; Reuland et al., 2014; Veldman et al., 2014), and none of them have been performed in

L. Ben Said et al. / International Journal of Food Microbiology 203 (2015) 86–92

the African continent. The aim of this study was to analyze the presence of ESBL-Eb in vegetable food samples collected from different farms and markets in Tunisia as well as in soil and treated water in the farm environment, and to characterize the recovered isolates. 2. Materials and methods 2.1. Sampling and bacterial identification One-hundred-fifty-four samples were included in this study: a) 109 samples collected from 18 different farms (49 of vegetables-food, 41 of soil and 19 of irrigation water); and b) 45 vegetable samples obtained from 13 different markets. All samples were obtained from different regions of Tunisia during January 2012–June 2013 and were tested for ESBL-Eb recovery. The type of vegetable-food samples tested in farms and markets was as follows (number of samples): lettuce (13), potato (11), onion (11), parsley (10), fennel (8), carrot (7), tomato (6), cereals (6), fruits (6), radish (5), cabbage, pepper and squash (3 each), and cucumber and olives (1 each). Samples of vegetable food, soil and irrigation water of each of the 18 tested farms were obtained at the same time. Samples were processed as follows: 30 g of vegetable or soil and 5 ml of irrigation water were homogenized with 270 ml of buffered peptonewater and incubated 24 h at 37 °C; after that, one ml was streaked onto MacConkey agar plates supplemented with cefotaxime (2 μg/ml) and incubated 24 h at 37 °C. Colonies with the morphology of Enterobacteriaceae (up to three) were selected per sample and screened for the ESBL phenotype by a double-disk test (CLSI, 2013), and they were identified by biochemical and molecular tests [PCR and sequencing of 16S rDNA (primers used: 16S-F, 5′-TTCTGCAG TCTAGAGGAGGT GWTCCAGGC-3′ and 16S-R, 5′-CCAGAGTTGATCMTGGCTCAG-3′)]. 2.2. Antibiotic susceptibility testing Susceptibility to 16 antibiotics (ampicillin, cefotaxime, cefoxitin, ceftazidime, chloramphenicol, ciprofloxacin, gentamicin, imipenem, nalidixic acid, streptomycin, sulphonamides, tetracycline, ticarcilline, tobramycin, trimethoprim, and trimethoprim-sulfamethoxazole) was carried out by disk-diffusion method (CLSI, 2013). E. coli ATCC 25922 was used as a control strain. 2.3. Characterization of beta-lactamase genes and genetic environment of blaCTX-M genes The genes encoding TEM, SHV, OXA-1, and CTX-M type betalactamases and the genetic environment of blaCTX-M genes were analyzed by PCR and sequencing in all ESBL-Eb isolates (Jouini et al., 2007). Nucleotides and their deduced amino acid sequences were compared with those included in the GenBank database as well as with those deposited at the website http://www.lahey.org/Studies, to confirm the specific type of beta-lactamase gene. The presence of genes associated with resistance to chloramphenicol [cmlA, floR and catB3], gentamicin [aac(3)-II, and aac(3)-IV], quinolones [qnr, qepA and aac(6′)-Ib-cr], streptomycin [strA and strB], sulphonamides [sul1, sul2, and sul3] and tetracycline [tet(A) and tet(B)], was determined by PCR (Sáenz et al., 2004).

87

2.5. Virulence genotyping E. coli strains were screened for the presence of virulence factors: fimA (encoding type 1 fimbriae), papG allele III (adhesin PapG class III), hlyA (hemolysin), cnf1 (cytotoxic necrotizing factor), papC (P fimbriae), aer (aerobactin iron uptake system), eae and bfp genes (encoding virulence factors often found in pathogenic E. coli (ExPEC) isolates) were tested by PCR (Ruiz et al., 2002). 2.6. Molecular typing of ESBL-Eb isolates Pulsed-field-gel-electrophoresis (PFGE) of XbaI-digested genomic DNA of ESBL-Eb isolates was performed as previously described (Sáenz et al., 2004), and DNA patterns were interpreted as previously recommended (Tenover et al., 1995). E. coli strains were characterized by Multilocus-sequence typing (MLST) by PCR amplification of the standard seven housekeeping loci (Tartof et al., 2005). All the amplicons were sequenced and compared with the sequences deposited in the MLST database (http://mlst. warwick.ac.uk/) to know the specific allele combination and the sequence type (ST). In addition, E. coli strains were assigned to the phylogenetic groups A, B1, B2, or D using a PCR strategy with specific primers for chuA, yjaA, and TspE4.C2 determinants as previously described (Clermont et al., 2000). 2.7. Plasmid detection, sizing and conjugal transfer Conjugation experiments were carried out in nutrient broth (NB) for all Eb-ESBL strains using rifampicin-resistant E. coli strain CSH26 (Lac-, rifampicin-resistant) or E. coli strain J53 (azide-resistant) as recipient strains. Luria–Bertani (LB) agar plates containing rifampicin (50 μg/L) and cefotaxime (5 μg/L) were used for recovery of transconjugants (TCs). Plasmids from donor and transconjugant strains were assigned to incompatibility groups by PCR-based replicon typing (PBRT) performed on total DNA as previously described (Carattoli et al., 2005). Eighteen pairs of primers were used to perform 5 multiplex- and 3 simplexPCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons. Positive controls were included in all PCRs. The number and size of plasmids in each ESBL-positive E. coli strain and their transconjugants were determined by genomic DNA digestion with S1 nuclease and subsequent PFGE analysis (Barton et al., 1995). 3. Results 3.1. Detection of ESBL-Eb in vegetable food, soil and irrigation water of farm origin ESBL-Eb were isolated in four of 49 vegetable samples (8.2%), in 3 of 41 soil samples (7.3%), and in 3 of 19 irrigation water samples (15.8%) (Table 1). In the positive samples, only one type of ESBL-Eb was obtained. The 10 ESBL-Eb detected in farm samples were identified as E. coli (n = 6), Enterobacter hormaechei (n = 2), Citrobacter freundii (n = 1), and Klebsiella pneumoniae (n = 1). All these ESBL-Eb were obtained in samples taken from 2 of the 18 studied farms (11.1%), and both farms (number 10 and 16) used treated wastewater for irrigation. 3.2. Detection of ESBL-Eb in vegetables of market origin

2.4. Detection and characterization of integrons The presence of intI1 and intI2 genes (encoding class 1 and class 2 integrases, respectively), and the 3′-conserved segment (3′-CS) (qacE Δ1-sul1 genes) of class 1 integrons was examined by PCR. The variable regions of class 1 integrons were characterized by PCR and sequencing in all intI1-positive isolates (Sáenz et al., 2004).

ESBL-Eb were isolated from 4 of 45 vegetable food samples of market origin (8.9%), which were obtained from three of the 13 tested markets (23.1%). In one tomato sample, two different ESBL-Eb isolates were obtained (E. coli and C. freundii) and in the remaining positive samples, only one type of ESBL-Eb was identified. The isolates recovered from market origin corresponded to: C. freundii (3 isolates) and E. coli (2

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L. Ben Said et al. / International Journal of Food Microbiology 203 (2015) 86–92

Table 1 ESBL-producing Enterobacteriaceae recovered from samples of vegetable food samples, as well as soil and irrigation water. Characteristics of ESBL-Eba detected

Source (number)

Type of samples (number)

Farms (n = 18)

Vegetable-food (n = 49) (37 greens, 6 cereals and 6 fruits)

Soil (n = 41)

Irrigation water (n = 19)

Markets (n = 13)

a b c

Vegetable-food (n = 45) (45 greens)

Species

Strain

Origin

E. coli E. coli E. hormaechei E. hormaechei – E. coli E. coli E. coli C. freundii K. pneumoniae E. coli E. coli C. freundii C. freundii E. coli C. freundii

C6246 C6249 C6250 C5913 – C6239 C5914 C5915 C5916 C5917 C6532 C6530 C5911 C5912 C6531 C6587

Oat (farm 10) Barley (farm 16) Barley (farm 10) Apricot (farm 10) – Farm 10 Farm 10 Farm 10 Farm 16 Farm 10 S19b Parsley (market 12) Fennel (market 3) Radish (market 3) Tomato (market 13)c Tomato (market 13)c

ESBL-Eb: Extended-spectrum beta-lactamase-producing Enterobacteriaceae. S19: sample of treated wastewater destined for farm irrigation. Strains recovered from the same sample.

isolates). The specific vegetables from which these ESBL-Eb isolates were obtained are indicated in Table 1. 3.3. Molecular characterization of genes encoding ESBL and their genetic environment Table 2 shows the results obtained with the collection of 15 ESBL-Eb recovered from vegetables, soil and irrigation water. ESBL genes identified among strains of different species were as follows (species/number of isolates): blaCTX-M-1 (E. coli/5); blaCTX-M-15 + blaTEM-1-b + blaOXA-1 (C. freundii/3; E. hormaechei/2); blaCTX-M-15+ blaOXA-1 (E. coli/2); blaSHV-12 + blaOXA-1 (C. freundii/1); blaCTX-M-14a (E. coli/1) and blaCTX-M-15+ blaTEM-1-b + blaOXA-1 + blaSHV-1 (K. pneumoniae/1). In order to evaluate which genetic mechanisms might be implicated in the mobilization of ESBL genetic determinants and consequently in their worldwide distribution, the region surrounding the blaCTX-M genes was analyzed by PCR and sequencing. The orf477 sequence was detected downstream of the blaCTX-M-15 gene in all eight strains containing this gene (orf477 was found to be disrupted by IS26 in strain K. pneumoniae C5917), and the ISEcp1 sequence was found in the upstream region in seven of them (this upstream region was unknown in one strain). The blaCTX-M-1 gene was found flanked in the five strains by orf477 (truncated by IS5 in three E. coli strains) and ISECp1 sequences. The ISEcp1 and IS903 were detected, respectively, upstream and downstream of the blaCTX-M-14a gene in one E. coli strain. Finally, IS26 was found upstream blaSHV-12 gene detected only in one strain. 3.4. Characterization of integrons and resistance mechanisms to non-betalactam antimicrobial agents Class 1 integrons were present in 11 of the 15 ESBL-Eb strains, but class 2 integrons were not identified in this collection. Only two different arrangements were detected, the first one characterized by the gene cassettes aadA5 + dfrA17 (confer resistance to streptomycin and trimethoprim, respectively), and the second arrangement was aadA1 found in three strains (implicated in streptomycin resistance). Six of the int1-positive ESBL-Eb strains lacked the qacE1-sul1 region characteristic of classic integrons. Table 2 shows the antibiotic resistance phenotypes and genotypes for the different ESBL-Eb. All strains (except E. coli C6530) showed multiresistance (including at least 3 families of antibiotics). A variety of different resistance genes were observed among our strains [antibiotic (number of resistant strains)/gene (number of strains)]: tetracycline

(8)/tet(A) (7)/tet(B) (1); streptomycin (9)/strA-strB (7); gentamicin (7)/aac(3′)-II (7); and chloramphenicol (5)/floR (1)/cmlA(1). The sul1 and sul2 genes were detected in 11 and 6 strains, respectively, and four of those strains showed both genes. All 8 blaCTX-M-15-positive strains carried the aac(6′)-1b-cr gene that affects to amikacin, tobramycin, kanamycin and ciprofloxacin and harbored bla OXA-1; Plasmid-mediated quinolone resistance genes of the qnr family were identified in 5 ESBLEb strains (qnr(B) in 3 C. freundii and one K. pneumoniae; qnr(S) in one C. freundii). 3.5. Molecular typing of ESBL-Eb strains Table 3 shows the diversity of the 8 ESBL-E. coli isolates of this study, in relation to the sequence types, phylogroups and PFGE-patterns. In this sense, the following sequence-types-associated phylogroups were detected among the ESBL-producing E. coli strains: a) CTX-M-1positive strains: ST58-B1 (n = 3), ST117/D (n = 1) and ST23/A (n = 1); b) CTX-M-15-positive strains: ST131-B2 (n = 1) and ST3496-D (n = 1); c) CTX-M-14a positive strain: ST10-A (n = 1). All E. coli isolates showed unrelated PFGE-patterns, except the strains C6246 and C6239 which presented indistinguishable patterns; these two strains contained the blaCTX-M-1 gene, were typed as ST58-B1, and were recovered from a cereal sample (oat) and a soil sample of the same farm (number 10) that used treated wastewater for irrigation. The three blaCTX-M-15-positive C. freundii isolates showed indistinguishable PFGE patterns and they were recovered from two vegetable samples of farm 3 and one irrigation water sample of farm 16 (treated wastewater). Moreover, the two blaCTX-M-15-positive E. hormaechei isolates, recovered vegetable samples of farm 10, presented unrelated PFGE patterns (Table 2). The fimA and aer virulence genes were detected in all ESBL-positive E. coli strains and eae gene was also found in four of these strains. The E. coli strain C5915 typed as ST131-B2 contained 5 of the 8 virulence genes studied (fimA-aer-hly-papC-cnf1) (Table 3). 3.6. Plasmid characterization and conjugal transfer of ESBL encoding genes The IncI1, IncF, IncFIB, IncFIA, IncK, IncY and IncB/O replicons were detected among our E. coli strains. The most prevalent replicons were IncI1, IncFIB and IncF that were found associated in six strains. From 1 to 2 plasmids were detected in each strain and the size ranged approximately from 63 Kb to 179 Kb. Table 3 shows the different replicon profiles detected and plasmid sizes. In the conjugation experiments, transfer of the ESBL phenotype was demonstrated in five out of eight

Table 2 Characteristics of the ESBL-Eb strains detected in vegetable-food, soil and irrigation water. ESBL genes and genetic environment

E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli C. freundii

Ec-A Ec-A Ec-B Ec-C Ec-D Ec-E Ec-F Ec-G Cf-A

ISEcp1c-blaCTX-M-1--orf477 ISEcp1c-blaCTX-M-1--orf477 ISEcp1c-blaCTX-M-1--orf477 ISEcp1-blaCTX-M-1-orf477 ISEcp1-blaCTX-M-1-orf477 ISEcp1-blaCTX-M-15-orf477 unknown-blaCTX-M-15-orf477 ISEcp1-blaCTX-M-14a -IS903 ISEcp1-blaCTX-M-15-orf477

+/− +/− +/− −/− −/− +/+ +/+ −/− +/+

– – – – – aadA5 + dfrA17 aadA5 + dfrA17 – aadA1

C5912 C. freundii

Cf-A

Radish (M-3)

ISEcp1-blaCTX-M-15-orf477

+/+

aadA1

C5916 C. freundii

Cf-A

Irrigation water (F-16)

ISEcp1-blaCTX-M-15-orf477

+/+

aadA1

C6587 C5913 C6250 C5917

Cf-B Eh-A Eh-B Kp-A

Tomato (M-13) Apricot (F-10) Barley (F-10) Irrigation water (F-10)

IS26-blaSHV-12 ISEcp1-blaCTX-M-15-orf477 ISEcp1-blaCTX-M-15-orf477 ISEcp1-blaCTX-M-15-orf477d

+/− −/− +/− +/−

– – – –

C6246 C6239 C6249 C6531 C6532 C5914 C5915 C6530 C5911

a b c d e f g h

Species

C. freundii E. hormaechei E. hormaechei K. pneumoniae

Oat (F-10) Soil (F-10) Barley (F-16) Tomato (M-13) Irrigation water (S19) Soil (F-10) Soil (F-10) Parsley (M-12) Fennel (M-3)

Class 1 integron Int1/3′-CS Integron structure

Resistance phenotype to non-β-lactam antibioticse

SXT–SUL–TET–STR–FOX SXT–SUL–TET–NAL–STR SXT–SUL–TET–STR–FOX SUL–TET–NAL SUL–TET–NAL SXT–SUL–TET–CIP–NAL–GEN–TOB–STR SXT–SUL–CIP–NAL–GEN–TOB–STR Susceptiblef SXT–SUL–CIP–NAL–GEN–TOB–STR–CHL

Other resistance genes detected outside integron

sul2, tet(A), strA/strB sul2, tet(A), strA/strB sul2, tet(A), strA/strB sul2, tet(A) sul2, tet(A) blaOXA-1,sul1, tet(B), aac(6′)-1b-cr, aac(3′)-II blaOXA-1,sul1, aac(6′)-1b-cr, aac(3′)-II – blaTEM-1-b, blaOXA-1, sul1, sul2, aac(6′)-1b-cr, qnrB, aac(3′)-II, strA/strB SXT–SUL–CIP–NAL–GEN–TOB–STR–CHL blaTEM-1-b, blaOXA-1, sul1, sul2, aac(6′)-1b-cr, qnrB, aac(3′)-II, strA/strB SXT–SUL–CIP–NAL–GEN–TOB–STR–CHL–FOX blaTEM-1-b, blaOXA-1, sul1, sul2, aac(6′)-1b-cr, qnrB, aac(3′)-II, strA/strB SXT–SUL–TET–CIP–NAL–CHL–FOX blaTEM-1-b, sul2, tet(A), qnrS, floR CIP–NAL–GEN–TOB–FOX blaTEM-1-b, blaOXA-1, aac(6′)-1b-cr, aac(3′)-II SXT–NAL–TOB–CHL blaTEM-1-b, blaOXA-1, aac(6′)-1b-cr, cmlA SXT–SUL–CIP–NAL–GEN–TOB–STR blaTEM-1-b, blaSHV-1, blaOXA-1,sul1, sul2, aac(6′)-1b-cr, qnrB, aac(3′)-II, strA/strB

Transference by conjugation of ESBL genes + + + + – + – – – +g – +h – – –

Ec: E. coli; Cf: C. freundii; Eh: E. hormaechei; Kp: K. pneumoniae. M: open-market; F: farm; S19: sample of treated wastewater prepared for farm irrigation. ISEcp1 disrupted by IS5 element. orf477 disrupted by IS26 element. Antibiotics: CHL: chloramphenicol; CIP: ciprofloxacin; FOX: cefoxitin; GEN: gentamicin; NAL: nalidixic acid; STR: streptomycin; SUL: sulphonamides; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; TOB: tobramycin. The isolate was susceptible for non-β-lactam agents. IncK replicon was detected in donor. No replicons were detected in donor or TC.

L. Ben Said et al. / International Journal of Food Microbiology 203 (2015) 86–92

Originb PFGE patterna

Strain

89

90

L. Ben Said et al. / International Journal of Food Microbiology 203 (2015) 86–92

Table 3 Molecular typing, PBRT plasmid typing and virulence factors of ESBL-producing E. coli isolated from vegetables and farm environment. Strain

ESBL type

Molecular typing MLST ST

ST complex

Phylo group

Virulence genes

Plasmids

Conjugative experiments and characteristics of TCc

Na Size (Kb)b Replicons

CTd ESBL Resistance Resistance genes (+/−) phenotype phenotypee + +

+ +

SUL–TET SXT–SUL

blaCTX-M-1, sul2, tet(A) I1 blaCTX-M-1, sul2, int1 I1

+

+

SXT–SUL

blaCTX-M-1, sul2, int1

I1

+

+

SXT–SUL

blaCTX-M-1, sul2, int1

I1

+

+

SXT–TET– GEN–TOB n.c n.c n.c

blaCTX-M-15, blaOXA-1, sul1, tet(B), aac(3′)-II n.c n.c n.c

I1, FIA, FIB, F n.c n.c n.c

C6531 CTX-M-1 C6246 CTX-M-1

117 none 58 155

D B1

fimA-aer fimA-aer-eae

2 2

97–179 97–160

C6249 CTX-M-1

58 155

B1

fimA-aer-eae

2

97–160

C6239 CTX-M-1

58 155

B1

fimA-aer-eae

2

97–160

D

fimA-aer

2

80–145

C5914 CTX-M-15 3496 none C5915 CTX-M-15 C6530 CTXM14a C6532 CTX-M-1 a b c d e f

131 none 10 10 23 23

B2 A A

fimA-aer-hly-papC-cnf1 1 fimA-aer 2 fimA-aer-eae 1

102 63–82 97

I1, FIB, F I1, FIA, FIB, F, K I1, FIA, FIB, F, K I1, FIA, FIB, F, K I1, FIA, FIB, F, Y FIA, F FIB, F, K, B/O I1, FIB, F, K

– –

n.c n.c n.c

f

Plasmid replicons

N: number of plasmids. Size of plasmids of donors. Those plasmids transferred by conjugation are indicated in bold and underlined numbers. TC: transconjugants; CT: transference by conjugation. G: gentamicin; SUL: sulphonamides; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; TM: tobramycin; n.c: non conjugative plasmids.

E. coli strains tested; however, transconjugants could not be recovered in the other three strains, despite two separate attempts. In four of the cases, the obtained transconjugants received the blaCTX-M-1 gene in addition to IncI replicon, which probably carried this ESBL gene. In addition, transconjugant from C5914 captured the blaCTX-M-15 gene in addition to different replicon plasmids (IncI1 and replicons of IncF family). Other resistance genes were co-transferred with ESBL gene (Table 3). The IncH1 replicon was detected in the two blaCTX-M-15-containing E. hormaechei isolates and the IncK replicon in the three blaCTX-M-15 C. freundii strains. All PCRs for replicon plasmid detection were negative in C. freundii C6587 and K. pneumoniae C5917. Transference by conjugation of ESBL encoding genes was tested in the 7 ESBL-positive C. freundii, E. hormaechei and K. pneumoniae strains but transconjugants were only obtained for C. freundii C5912 (receptor E. coli J53) and C. freundii C6587 (receptor E. coli CSH26) (Table 2). Transconjugant TC5912 acquired the ESBL phenotype as well as resistance to trimethoprim-sulfamethoxazole and streptomycin. Transconjugant TC6587 only acquired the ESBL phenotype but not other resistance markers of donor strain. 4. Discussion It is of interest to highlight that ESBL-Eb were detected in two of the 18 farms included in this study (farms 10 and 16), which corresponded to those that used treated wastewater for irrigation. To our knowledge, there was no common source in the irrigation water used in both farms. Moreover, ESBL-Eb were also detected in an additional treated wastewater sample prepared for farm irrigation (S9) analyzed in this study. In the case of farm 10, ESBL-Eb were detected in different vegetable food samples (E. coli and E. hormaechei carrying blaCTX-M-15 gene), soil (E. coli carrying blaCTX-M-1 and blaCTX-M-15 genes), and irrigation water (K. pneumoniae carrying blaCTX-M-15 gene). In the same way, different ESBL-Eb were detected in vegetables (E. coli carrying blaCTX-M-1) and irrigation water (C. freundii carrying blaCTX-M-15) taken in farm 16. In this study, it was observed a high heterogeneity in the frequency of ESBL-Eb at the farm level, as no ESBL-Eb were detected in the remaining 16 farms. Many factors could be behind these differences, but the type of water used (treated wastewater or not) and the potential use of manure as fertilizers could be important in this case. Some authors have already reported the frequent detection of ESBL-Eb in wastewater treatment plants (Alouache et al., 2014) and potential transfer of antibiotic resistant bacteria through the treated wastewater used in agriculture (Pignato et al., 2009), although others did not found so clear relationship (Negreanu et al., 2012). The application of organic manure to

agricultural fields has also been identified as a route of dissemination of multidrug-resistant bacteria (Boehme et al., 2004). ESBL-Eb that contaminates vegetables can be transmitted to human consumers via the food chain. In the present work, ESBL-Eb were detected in 4 of 45 vegetable samples of market origin tested and in 3 of 13 markets. ESBL-Eb positive isolates were also detected among vegetables samples in the Netherlands (Reuland et al., 2014). It is of interest to remark that vegetables containing ESBL-Eb of market origin are normally eaten uncooked, and the possibility of transference to humans cannot be discarded. Different authors also indicate the potential problems derived from these uncooked food samples containing antibiotic resistant bacteria (Blaak et al., 2014; Hamilton-Miller and Shah, 2001; Schwaiger et al., 2011; Veldman et al., 2014). In fact, foodborne outbreaks due to ESBL-positive E. coli isolates related to vegetables have been previously reported (Buchholz et al., 2011; King et al., 2012). The variety of Enterobacteriaceae species and resistance genes detected among the ESBL-Eb in this study (E. coli, C. freundii, E. hormaechei, and K. pneumoniae) indicates the potential transference of plasmids carrying ESBL in bacteria of different ecosystems, probably in the intestinal tract of humans or animals and later in the environment. In our study, it was demonstrated that more than half of ESBL-E. coli recovered could transfer the ESBL genes by conjugation to other bacteria (with high frequency of conjugation, data not shown). Horizontal transfer of antibiotic resistant genes between soil and human pathogens has been previously analyzed and demonstrated (Forsberg et al., 2012). The transference of the blaCTX-M-1 gene in 4 of our E. coli strains, was associated with the acquisition of the IncI1 replicon plasmid, as has already been reported by other authors in E. coli isolates of human and animal origins (Ben Sallem et al., 2014; Mnif et al., 2013). Transference of ESBL genes by conjugation was also demonstrated in two of the C. freundii isolates when E. coli was used as receptor strain, although it could be associated to a non-typeable plasmid. ESBL-producing E. hormaechei isolates are unfrequently reported, although in the last years there are several reports related with the detection of ESBL- or carbapenemase-producing E. hormaechei isolates at the hospital level (Carvalho-Assef et al., 2014). Two unrelated CTX-M15 producers E. hormaechei were detected in this study in different vegetables of one farm. The role of vegetables in the spread of this multiresistant microorganism should be evaluated in the future comparing isolates of food and human origin. The ESBL variants detected in our study (CTX-M-15, CTX-M-14, CTXM-1 and SHV-12) correspond to the most frequently ESBL variants previously found in Tunisia among clinical E. coli or E. cloacae isolates

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(CTX-M-15, CTX-M-14, SHV-12) (Ben Slama et al., 2011; Jouini et al., 2010; Lahlaoui et al., 2012; Mnif et al., 2013) and in E. coli from food, food-producing animals, and healthy humans (CTX-M-1, CTX-M-14) (Ben Sallem et al., 2011; Ben Sallem et al., 2012; Jouini et al., 2007). In this sense, it seems that contamination by ESBL-Eb of vegetable samples and farm environments could be driven by human and animal sources. The potential implication of wastewater used for irrigation cannot be discarded as a route for the dissemination of these antibiotic resistant bacteria. Additionally, it has been reported an increase in the carriage of antibiotic resistance genes in soil along the years (Knapp et al., 2010), highlighting the role of this element as a reservoir of antibiotic resistant bacteria and genes. Previous studies have reported the isolation of Enterobacteriaceae harboring CTX-M-14 and CTX-M-15 ESBL in fresh culinary herbs from Southeast Asia, highlighting the potential human health risk associated with their consumption (Veldman et al., 2014). In addition, a recent report carried out in raw vegetables from the Netherlands refers the detection of the same ESBL variants (CTX-M-15, CTX-M-14, CTX-M-1 and SHV-12) as the one detected in our study (Reuland et al., 2014). Most of our ESBL-Eb showed a multiresistance phenotype (except the strain C6530 harboring blaCTX-M-14a gene), and carried integrons, being this character typical from ESBL-producing bacteria in other studies (Schmiedel et al., 2014). The high diversity of clones detected among ESBL-positive E. coli (6 different ST among 8 strains) indicates that the dissemination of this resistance is not associated to the spread of an unique clone but rather to the dissemination of plasmids containing these genes, as has already been reported (Ben Sallem et al., 2014). It is of relevance the detection of the ST131-B2 clone, carrying the CTX-M-15 gene and containing multiple virulence and resistance genes, in an E. coli strain recovered from soil at the farm environment. This clone is widely disseminated in Tunisian hospitals, as well as in other hospitals all around the word (Banerjee et al., 2013; Seiffert et al., 2013). It seems that clones and genetic variants of ESBLs are not restricted to a unique ecosystem but a continuous flow is occurring. 5. Conclusion Food vegetables can be contaminated with ESBL-Eb, especially those coming from farms that use treated wastewater for irrigation, and these resistant bacteria might be transmitted to human through the food chain, particularly if these products are eaten uncooked. Future studies should be carried out to evaluate the real risk for human health of this potential transmission linked to the consumption of food vegetables. Acknowledgment This work was partially supported by Project SAF2012-35474 from the Ministerio de Economía y Competitividad of Spain and the Fondo Europeo de Desarrollo Regional (FEDER), and by project from the Tunisian Ministry of Higher Education, Scientific Research and Information and Communication Technologies. We thank A. Caratolli for providing us with positive controls for PBRT testing. Carla Andrea Alonso has a predoctoral fellowship FPI from the Ministerio de Economía y Competitividad of Spain. References Alouache, S., Estepa, V., Messai, Y., Ruiz, E., Torres, C., Bakour, R., 2014. Characterization of ESBLs and associated quinolone resistance in Escherichia coli and Klebsiella pneumoniae isolates from an urban wastewater treatment plant in Algeria. Microb. Drug Resist. 20, 30–38. Bain, R., Cronk, R., Hossain, R., Bonjour, S., Onda, K., Wright, J., Yang, H., Slaymaker, T., Hunter, P., Prüss-Ustün, A., Bartram, J., 2014. Global assessment of exposure to faecal contamination through drinking water based on a systematic review. Trop. Med. Int. Health 19, 917–927.

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