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International Journal of Antimicrobial Agents 25 (2005) 308312

Acquired sulphonamide resistance genes in faecal Escherichia colifrom healthy children in Bolivia and Peru

Beronica Infante a, 1, Malin Grape a, Mattias Larsson b, Charlotte Kristiansson b,Lucia Pallecchi c, Gian Maria Rossolini c, Goran Kronvall a,

a Microbiology and Tumor Biology Centre (MTC), Clinical Microbiology, Karolinska Institute, SE-17176 Stockholm, Swedenb Department of Public Health Science, Division of International Health (IHCAR), Karolinska Institute, SE-17176 Stockholm, Sweden

c Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Universit` a di Siena, I-53100 Siena, Italy

Received 25 October 2004; accepted 9 December 2004

Abstract

Antimicrobial resistance and sulphonamide resistance determinants were studied in 20 co-trimoxazole resistant Escherichia coli in faecalsamples from healthy children in Bolivia and Peru. Methods used were disc diffusion susceptibility tests, PCR, sequence analysis and plasmidconjugation assays. All isolates but one were resistant to at least two different classes of antimicrobials; 19 isolates also carried at least onesul -gene. The most frequent gene was sul2 followed by sul1 and sul3 , which was detected in one isolate. This is the rst observation of sul3on the American continent. In conclusion, the high prevalence of sul -genes in this material of faecal commensal E. coli isolates points to apotential role of the faecal ora in the emergence and spread of antimicrobial resistance. 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: Sulphonamide resistance; Co-trimoxazole; sul; Integron; E. coli

1. Introduction

Since their introduction in clinical practice in 1935,sulphonamides have been widely used in human and veteri-nary medicine [1,2]. However, to overcome the rapid emer-gence and spread of resistance that has strongly limited theuse of these inexpensive antibacterial drugs, sulphonamideshave generally been combined with diaminopyrimidines,such as trimethoprim [1,2]. In Escherichia coli , acquired re-sistance to sulphonamides can result either from mutations

in the chromosomal dihydropteroate synthase (DHPS) gene( folP ), which decrease DHPS afnity for the sulphonamideinhibitors or, more frequently, from the acquisition of sul-type genes that encode DHPSs with reduced afnity forsulphonamides [1,2]. Three such determinants have been

Corresponding author. Tel.: +46 8 51774910; fax: +46 8 308099. E-mail address: goran.kronvall@labmed.ki.se (G. Kronvall).

1 Present address: Instituto de Medicina Tropical Alexander von Hum-boldt, Universidad Peruana, Cayetano Heredia, A.P. 4314 Lima 100, Peru.

characterized, sul1 , sul2 and sul3 . The rst one, sul1 , is usu-ally associated with class 1 integrons, being part of the so-called3 -conserved-segment (3 -CS) of these elements [25] .The sul2 gene was found to be mediated by plasmids of var-ious types, whilst sul3 is a novel sul -type allele, encoding amore divergent DHPS lineage [2,3,6,7] . This gene was re-cently identied in E. coli isolates from pigs in Switzerland,and was found to be a quite common resistance determinantamong sulphonamide-resistant E. coli from cattle, swine andpoultry in Germany [1,8]. In Sweden, sul3 has recently been

detected in two urinary E. coli isolates from human sources[9].In recentyears, the potential role of the commensalmicro-

biotafor theemergenceand spread of antimicrobial resistancehas beenuniversally acknowledged [10]. Some species of thecommensal microbiota, such as faecal E. coli , have been ex-ploited as sensitive indicators in surveillance of antimicrobialresistance [1115] .

In this paper, we describe the characterization of acquiredsulphonamide resistance determinants of the sul -type in fae-

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cal E. coli of healthy children living in urban areas of Boliviaand Peru. The nding of the recently described sul3 deter-minant in this context represents the rst description of thisresistance gene in the Americas.

2. Materials and methods

2.1. Bacterial isolates

The E. coli investigated in this study were isolates col-lected during the pilot study of the ANTRES project, a col-laborative researchproject dealing withantimicrobial useandresistance in twoLatin American countries, Bolivia andPeru.In that pilot study, faecal swabs were obtained from healthychildren aged 672 months from four different urban areas(Camiri and Villa Montes in Bolivia, and Yurimaguas andMoyobamba in Peru), and E. coli resistant to various antimi-crobial agents were isolated by a rapid screening method,essentially as described previously [12]. Isolates resistant toco-trimoxazole were detected in the samples from 58 of 70subjects (83%), with no signicant differences between thesampled areas. Of these isolates, 20 were randomly selectedfor further analysis of acquired sulphonamide resistance de-terminants.

2.2. In vitro susceptibility testing

Antimicrobial susceptibility testing was performed by thediscdiffusion method on Iso-SensitestAgar(Oxoid Ltd., Bas-ingstoke, UK) with discs from Oxoid using the methodology

described by the Swedish Reference Group for Antibiotics(SRGA, http://www.srga.org ). Results of susceptibility test-ing were interpreted according to the criteria established bySRGA (http://www.srga.org ) [16,17] . E. coli ATCC 25922was used for quality control purposes in susceptibility test-ing.

2.3. Species identication

Identication of E. coli isolates was always conrmedusing a series of biochemical tests (Voges-Proskauer, man-nitol fermentation/gas production and production of beta-galoactosidase, lysine decarboxylase, ornithine decarboxy-lase, urease and indole) using numerical identication proce-dures [18]. E. coli ATCC 25922 was used as a quality controlorganism.

2.4. Molecular analysis techniques

Basic procedures for DNA extraction, analysis and ma-nipulation were performed as described by Sambrook andRussel [19]. PCR amplication of sul-type determinants wascarried out with primers designed for specic amplicationof sul1 , sul2 and sul3 genes, as described previously [1,9].Amplication of the entire sul3 gene for sequencing was car-

ried out with primers ORF1-fw (5 -GAA GAA TGG GAAGCT GTT AG-3 ) and SUL3-rev (5 -CTA AGT CAA GTACGC CAA CA-3 ), designed for this study on the sul3 ank-ing regions of plasmid pVP440 [1]. PCR amplication of class 1 integrase gene ( intI1 ) was carried out with primersIntI1-1 (5 -TGC GGG TCA AGG ATG TGG ATT T3 ) and

IntI1-2 (5 -CAA CAC ATG CGT GTA AAT-3 ), as describedpreviously [20]. PCR amplication of the variable region of class 1 integrons was carried out with primers Igr1-1 (5 -GCT CTA GAC CGA AAC CTT GCG CTC-3 ) and Igr1-2(5 -GGA ATT CAT GAT ATA TCT CCC AAT TTG T-3 ),as described previously [21]. PCR was performed with crudebacterial lysates obtained by boiling bacterial overnight cul-tures in distilled water for 3min, followed by centrifugationat 14,000 rpm for 1 min. Clinical E. coli isolates carrying thesul3 gene and E. coli C600, containing the pMS128 plasmidcarrying an integron of class 1 or R483 with an integron of class 2 were used as positive controls [9,20]. Plasmid DNAwas extracted by the alkaline lysis method [19]. Southernblot hybridization was carried out directly on dried gels asdescribed previously with a sul3 probe generated with thesul3 specic primers (see above) [22]. Primers were labelledwith 32P by the random-priming technique. Direct sequenc-ing of PCR products was carried out as described previously[21]. Additional analysis of nucleotide sequences was per-formed with the programme Sequencher 3.0 (Gene CodesCorporation, Ann Arbor, MI, USA). Comparative analysis of nucleotide sequences was done using the advanced BLASTsearch programme within the QBLAST system at the NCBIweb site (http://www.ncbi.nlm.nih.gov ).

2.5. Conjugation assays

Conjugal transfer of sulphonamide resistance determi-nants was assayed in MuellerHinton (MH) agar plates us-ing E. coli MKD-135 ( argH rpoB18 rpoB19 recA rpsL )as the recipient, and an initial donor/recipient ratio of 0.1.Mating plates were incubated at 30 C for 14 h. Transcon- jugants were selected on MH agar containing rifampicin(400 g/ml) plus sulphamethoxazole (200 g/ml). Under theabove conditions, the detection sensitivity of the assay was 5 10 8 transconjugant/recipient.

3. Results and discussion

3.1. Characterization of sul-type genes insulphonamide-resistant faecal E. coli isolates

Twenty E. coli isolates, randomly selected from 58 co-trimoxazole resistant isolates from faecal swabs of healthychildren living in various urban areas of Latin America, weresubjected to susceptibility testing and molecular characteri-zation for the presence of sul -type resistance determinants.

In vitro susceptibility testing was carried out with ampi-cillin (AMP), piperacillin/tazobactam (TZP), cefuroxime

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Table 1Resistance phenotype, sul-type determinants and class 1 integrons in the 20 co-trimoxazole resistant faecal E. coli isolates

Isolate Other resistance sul-genes Class 1 integrons

sul1 sul2 sul3 intI1 Variable region (kb) Gene cassettes

1 +2 CHL, TET +

3 TET, GEN + +4 TET +5 AMP, CHL, TET + + + 1.8 dfr1 , aadA16 AMP, CHL, TET + + + 1.8 dfr1 , aadA17 AMP, CHL, TET + + 2.8 dfr1 , catB3 , aacA48 CHL, TET, GEN + + +9 AMP, CHL, TET + + 1.8 dfr1 , aadA1

10 CHL, TET +11 AMP +12 TET + + 1.2 aadA113 AMP, CHL, TET +14 AMP +15 AMP, TET +16 AMP, CHL, TET17 AMP +

18 AMP, CHL, TET + +19 AMP, CHL + + 1.2 aadA120 AMP, CHL, TET + +

CHL: chloramphenicol, TET: tetracycline, GEN: gentamicin and AMP: ampicillin.

(CXM), cefotaxime (CTX), ceftazidime (CAZ), imipenem(IPM), loracarbef (LOR), gentamicin (GEN), netilmicin(NET), amikacin (AMK), tetracycline (TET) and chloram-phenicol (CHL). All but one of the studied isolates were re-sistant to antimicrobials of at least two of the different classestested and as many as eight isolates showed the same resis-

tance phenotype with antimicrobial resistance to ampicillin,chloramphenicol and tetracycline in addition to trimethoprimand sulphonamides ( Table 1 ).

Screening for sul -genes by PCR revealed the presence of at least one sul-type determinant in 19/20 isolates (95%).In 15 isolates, a single sul -type resistance determinant wasfound (sul1 in 4 and sul2 in 11 isolates, respectively), whilstin 4 isolates 2 different sul-type determinants were found(sul1 and sul2 in 3 isolates, and sul2 and sul3 in 1 isolate,respectively) ( Table 1 ). Overall, the prevalence of sul1 , sul2and sul3 determinants in the studied isolates were 35, 75 and5%, respectively. Sequence analysis of the sul3 gene showedthat it was identical to that previously described in Europeanisolates [1,8,9].

Transferability of the sul3 determinant was assayed inmating experiments using E. coli MKD-135 as recipient,and sulphamethoxazole plus rifampicin for selection of transconjugants. Sulphonamide resistance was transferred ata frequency of about 1 10 4 transconjugants/recipient. Of three randomly selected transconjugants, all exhibited re-sistance to sulphamethoxazole, and chloramphenicol, whilstthe susceptibility to trimethoprim, sulphamethoxazole, gen-tamicin and tetracycline was not affected (the other resis-tance traits of the donor). PCR analysis, carried out on thethree transconjugants, conrmed the presence of the sul3

determinant in all of them. Analysis of the plasmid con-tent of the three transconjugants revealed, in all cases, thepresence of a plasmid of approximately 100 kb, on the ba-sis of restriction mapping with Pst I. In a Southern blot hy-bridization, the sul3 probe recognized a 4.2 kb Pst I fragment(Fig. 1).

As observed previously, sul-type genes were extremelycommon in sulphonamide-resistant E. coli isolates, with

Fig.1. (A) Agarosegel electrophoresis of a plasmidDNA preparation fromatransconjugant obtained from the sul3 -harbouring isolate, either undigested(lane 1) or digested with Pst I (lane 2). (B) Results of Southern blot analysisbyusinga sul3 probe. Plasmids fromother transconjugants showed identicalrestriction proles. DNA size standard (in kilobases) are shown on the left.

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