International Journal of Antimicrobial Agents 25 (2005) 308312

Acquired sulphonamide resistance genes in faecal Escherichia coli from healthy children in Bolivia and Peru
Ber onica Infante a,1 , Malin Grape a , Mattias Larsson b , Charlotte Kristiansson b , Lucia Pallecchi c , Gian Maria Rossolini c , G oran Kronvall a,
c a Microbiology and Tumor Biology Centre (MTC), Clinical Microbiology, Karolinska Institute, SE-17176 Stockholm, Sweden Department of Public Health Science, Division of International Health (IHCAR), Karolinska Institute, SE-17176 Stockholm, Sweden Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Universit` a di Siena, I-53100 Siena, Italy b

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 faecal samples from healthy children in Bolivia and Peru. Methods used were disc diffusion susceptibility tests, PCR, sequence analysis and plasmid conjugation assays. All isolates but one were resistant to at least two different classes of antimicrobials; 19 isolates also carried at least one sul-gene. The most frequent gene was sul2 followed by sul1 and sul3, which was detected in one isolate. This is the rst observation of sul3 on the American continent. In conclusion, the high prevalence of sul-genes in this material of faecal commensal E. coli isolates points to a potential 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 veterinary medicine [1,2]. However, to overcome the rapid emergence and spread of resistance that has strongly limited the use of these inexpensive antibacterial drugs, sulphonamides have generally been combined with diaminopyrimidines, such as trimethoprim [1,2]. In Escherichia coli, acquired resistance to sulphonamides can result either from mutations in the chromosomal dihydropteroate synthase (DHPS) gene (folP), which decrease DHPS afnity for the sulphonamide inhibitors or, more frequently, from the acquisition of sultype genes that encode DHPSs with reduced afnity for sulphonamides [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 Humboldt, Universidad Peruana, Cayetano Heredia, A.P. 4314 Lima 100, Peru.

characterized, sul1, sul2 and sul3. The rst one, sul1, is usually associated with class 1 integrons, being part of the socalled 3 -conserved-segment (3 -CS) of these elements [25]. The sul2 gene was found to be mediated by plasmids of various types, whilst sul3 is a novel sul-type allele, encoding a more divergent DHPS lineage [2,3,6,7]. This gene was recently identied in E. coli isolates from pigs in Switzerland, and was found to be a quite common resistance determinant among sulphonamide-resistant E. coli from cattle, swine and poultry in Germany [1,8]. In Sweden, sul3 has recently been detected in two urinary E. coli isolates from human sources [9]. In recent years, the potential role of the commensal microbiota for the emergence and spread of antimicrobial resistance has been universally acknowledged [10]. Some species of the commensal microbiota, such as faecal E. coli, have been exploited as sensitive indicators in surveillance of antimicrobial resistance [1115]. In this paper, we describe the characterization of acquired sulphonamide resistance determinants of the sul-type in fae-

0924-8579/$ see front matter 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.12.004

B. Infante et al. / International Journal of Antimicrobial Agents 25 (2005) 308312

309

cal E. coli of healthy children living in urban areas of Bolivia and Peru. The nding of the recently described sul3 determinant in this context represents the rst description of this resistance gene in the Americas.

2. Materials and methods 2.1. Bacterial isolates The E. coli investigated in this study were isolates collected during the pilot study of the ANTRES project, a collaborative research project dealing with antimicrobial use and resistance in two Latin American countries, Bolivia and Peru. In that pilot study, faecal swabs were obtained from healthy children aged 672 months from four different urban areas (Camiri and Villa Montes in Bolivia, and Yurimaguas and Moyobamba in Peru), and E. coli resistant to various antimicrobial agents were isolated by a rapid screening method, essentially as described previously [12]. Isolates resistant to co-trimoxazole were detected in the samples from 58 of 70 subjects (83%), with no signicant differences between the sampled areas. Of these isolates, 20 were randomly selected for further analysis of acquired sulphonamide resistance determinants. 2.2. In vitro susceptibility testing Antimicrobial susceptibility testing was performed by the disc diffusion method on Iso-Sensitest Agar (Oxoid Ltd., Basingstoke, UK) with discs from Oxoid using the methodology described by the Swedish Reference Group for Antibiotics (SRGA, http://www.srga.org). Results of susceptibility testing were interpreted according to the criteria established by SRGA (http://www.srga.org) [16,17]. E. coli ATCC 25922 was used for quality control purposes in susceptibility testing. 2.3. Species identication Identication of E. coli isolates was always conrmed using a series of biochemical tests (Voges-Proskauer, mannitol fermentation/gas production and production of betagaloactosidase, lysine decarboxylase, ornithine decarboxylase, urease and indole) using numerical identication procedures [18]. E. coli ATCC 25922 was used as a quality control organism. 2.4. Molecular analysis techniques Basic procedures for DNA extraction, analysis and manipulation were performed as described by Sambrook and Russel [19]. PCR amplication of sul-type determinants was carried out with primers designed for specic amplication of 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 GAA GCT GTT AG-3 ) and SUL3-rev (5 -CTA AGT CAA GTA CGC CAA CA-3 ), designed for this study on the sul3 anking regions of plasmid pVP440 [1]. PCR amplication of class 1 integrase gene (intI1) was carried out with primers IntI1-1 (5 -TGC GGG TCA AGG ATG TGG ATT T3 ) and IntI1-2 (5 -CAA CAC ATG CGT GTA AAT-3 ), as described previously [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 crude bacterial lysates obtained by boiling bacterial overnight cultures in distilled water for 3 min, followed by centrifugation at 14,000 rpm for 1 min. Clinical E. coli isolates carrying the sul3 gene and E. coli C600, containing the pMS128 plasmid carrying an integron of class 1 or R483 with an integron of class 2 were used as positive controls [9,20]. Plasmid DNA was extracted by the alkaline lysis method [19]. Southern blot hybridization was carried out directly on dried gels as described previously with a sul3 probe generated with the sul3 specic primers (see above) [22]. Primers were labelled with 32 P by the random-priming technique. Direct sequencing of PCR products was carried out as described previously [21]. Additional analysis of nucleotide sequences was performed with the programme Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI, USA). Comparative analysis of nucleotide sequences was done using the advanced BLAST search programme within the QBLAST system at the NCBI web site (http://www.ncbi.nlm.nih.gov). 2.5. Conjugation assays Conjugal transfer of sulphonamide resistance determinants was assayed in MuellerHinton (MH) agar plates using 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. Transconjugants were selected on MH agar containing rifampicin (400 g/ml) plus sulphamethoxazole (200 g/ml). Under the above conditions, the detection sensitivity of the assay was 5 108 transconjugant/recipient. 3. Results and discussion 3.1. Characterization of sul-type genes in sulphonamide-resistant faecal E. coli isolates Twenty E. coli isolates, randomly selected from 58 cotrimoxazole resistant isolates from faecal swabs of healthy children living in various urban areas of Latin America, were subjected to susceptibility testing and molecular characterization for the presence of sul-type resistance determinants. In vitro susceptibility testing was carried out with ampicillin (AMP), piperacillin/tazobactam (TZP), cefuroxime

310

B. Infante et al. / International Journal of Antimicrobial Agents 25 (2005) 308312

Table 1 Resistance phenotype, sul-type determinants and class 1 integrons in the 20 co-trimoxazole resistant faecal E. coli isolates Isolate Other resistance sul-genes sul1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CHL, TET TET, GEN TET AMP, CHL, TET AMP, CHL, TET AMP, CHL, TET CHL, TET, GEN AMP, CHL, TET CHL, TET AMP TET AMP, CHL, TET AMP AMP, TET AMP, CHL, TET AMP AMP, CHL, TET AMP, CHL AMP, CHL, TET sul2 + + + + + + + + + + + + + + + + + + + 1.2 aadA1 + sul3 Class 1 integrons intI1 Variable region (kb) Gene cassettes

+ + + +

+ + + + +

1.8 1.8 2.8 1.8

dfr1, aadA1 dfr1, aadA1 dfr1, catB3, aacA4 dfr1, aadA1

+ + +

1.2

aadA1

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 chloramphenicol (CHL). All but one of the studied isolates were resistant to antimicrobials of at least two of the different classes tested and as many as eight isolates showed the same resistance phenotype with antimicrobial resistance to ampicillin, chloramphenicol and tetracycline in addition to trimethoprim and 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 was found (sul1 in 4 and sul2 in 11 isolates, respectively), whilst in 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, sul2 and sul3 determinants in the studied isolates were 35, 75 and 5%, respectively. Sequence analysis of the sul3 gene showed that it was identical to that previously described in European isolates [1,8,9]. Transferability of the sul3 determinant was assayed in mating experiments using E. coli MKD-135 as recipient, and sulphamethoxazole plus rifampicin for selection of transconjugants. Sulphonamide resistance was transferred at a frequency of about 1 104 transconjugants/recipient. Of three randomly selected transconjugants, all exhibited resistance to sulphamethoxazole, and chloramphenicol, whilst the susceptibility to trimethoprim, sulphamethoxazole, gentamicin and tetracycline was not affected (the other resistance traits of the donor). PCR analysis, carried out on the three transconjugants, conrmed the presence of the sul3

determinant in all of them. Analysis of the plasmid content of the three transconjugants revealed, in all cases, the presence of a plasmid of approximately 100 kb, on the basis of restriction mapping with PstI. In a Southern blot hybridization, the sul3 probe recognized a 4.2 kb PstI fragment (Fig. 1). As observed previously, sul-type genes were extremely common in sulphonamide-resistant E. coli isolates, with

Fig. 1. (A) Agarose gel electrophoresis of a plasmid DNA preparation from a transconjugant obtained from the sul3-harbouring isolate, either undigested (lane 1) or digested with PstI (lane 2). (B) Results of Southern blot analysis by using a sul3 probe. Plasmids from other transconjugants showed identical restriction proles. DNA size standard (in kilobases) are shown on the left.

B. Infante et al. / International Journal of Antimicrobial Agents 25 (2005) 308312

311

sul2 being the most prevalent and sul3 the least common [1,6,8,9,23]. This is the rst description of a sul3 determinant in America, revealing that its occurrence can be widespread also outside Europe. 3.2. Presence of class 1 integrons in the sulphonamide-resistant E. coli isolates The 20 E. coli isolates were also screened for the presence of class 1 integrons, which are known to be associated with the sul1 gene [4,5]. Overall, 9 of the 20 isolates were positive for the presence of the intI1 gene and from 6 of these isolates it was possible to amplify the integron variable region with primers designed to be specic for the intI1 and qacE 1 genes. In those cases, amplication products obtained from the variable regions showed molecular sizes of 1.2 kb in two isolates, 1.8 kb in three isolates and 2.8 kb in one isolate (Table 1). Sequence analysis revealed that: (a) the 1.2 kb amplication products carried an aadA1 gene cassette identical to that described in GenBank accession number AF071413, encoding an aminoglycoside adenylyl transferase [24]; (b) the 1.8 kb amplication products carried two gene cassettes, including a dfr1 cassette identical to that published in accession number AY007807, encoding a dihydrofolate reductase (DHFR) and an aadA1 cassette identical to that present in the smaller integron; (c) the 2.8 kb amplication product carried three gene cassettes, including a dfr1 cassette identical to that in accession number AY260546 whose DHFR product was 98% identical to that encoded by the 1.8kb integron, a catB3 cassette identical to that published in accession number AY259086 encoding a chloramphenicol acetyl-transferase and an aacA4 cassette identical to that in accession no. AF445082 encoding an aminoglycoside acetyltransferase (Table 1). All six isolates positive for class 1 integrons from which it was possible to amplify the variable region also possessed the sul1 gene, which is consistent with the presence of sulassociated class 1 integrons [4]. On the other hand, the three class 1 integron-positive isolates from which it was not possible to amplify the variable region using primers Igr1-1 and Igr1-2 (designed on the 5 - and 3 -CS of class 1 integrons), were sul1 negative suggesting the presence of a class 1 integron lacking parts of or the entire 3 sequence typical for this class of integrons. Finally, in isolate no. 3 (see Table 1); the presence of sul1 was apparently independent of the presence of a class 1 integron. Similar ndings have been reported earlier and could either reect the presence of a sul1 determinant located in a different genetic context, or a sequence change that has involved the 5 -CS of a sul1-associated integron [6,20,25]. 3.3. Conclusions and comments In the present study, E. coli resistant to co-trimoxazole were found to be common (83%) in the commensal microbiota of healthy children living in urban areas of Bolivia and

Peru. In these isolates, sul-type determinants were found to be almost universally present. Even in a small sample, all the three known types of sul-genes were found to be represented, suggesting a widespread distribution of these resistance determinants. Of the three known variants, sul2 was the one most frequently detected, similarly to what had been observed in previous studies carried out with human and animal isolates [6,8,9,23,25]. The high prevalence of sul-genes among the intestinal E. coli suggests that commensals could represent an important reservoir of these resistance determinants. Acknowledgements This work was supported by a grant from the European Commission (ANTRES project, INCO-DEV Contract no. ICA4-CT-2001-10014), and by a grant from AFA Health foundation, Stockholm, Sweden. B.I. is a recipient of a Marie Curie Scholarship, within the ANTRES project. We would like to thank Inga Karlsson for technical support in the laboratory. References
[1] Perreten V, Boerlin P. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother 2003;47:116972. [2] Sk old O. Sulfonamide resistance: mechanisms and trends. Drug Resist Updat 2000;3:15560. [3] Huovinen P, Sundstr om L, Swedberg G, Sk old O. Trimethoprim and sulfonamide resistance. Antimicrob Agents Chemother 1995;39:27989. [4] Recchia GD, Hall RM. Gene cassettes: a new class of mobile element. Microbiology 1995;141:301527. [5] Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding ite-specic gene-integration functions: integrons. Mol Microbiol 1989;3:166983. [6] Enne VI, Livermore DM, Stephens P, Hall LM. Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. Lancet 2001;357:13258. [7] Enne VI, Bennett PM, Livermore DM, Hall LM. Enhancement of host tness by the sul2-coding plasmid p9123 in the absence of selective pressure. J Antimicrob Chemother 2004;53:95863. [8] Guerra B, Junker E, Schroeter A, Malorny B, Lehmann S, Helmuth R. Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J Antimicrob Chemother 2003;52:48992. [9] Grape M, Sundstr om L, Kronvall G. Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources. J Antimicrob Chemother 2003;52:10224. [10] Alliance for the Prudent Use of Antibiotics (APUA), Reservoirs of Antibiotic Resistance (ROAR). Commensal bacteria are reservoirs of resistance. http://www.tufts.edu/med/apua/ROAR/projects.htm. [11] Bartoloni A, Cutts F, Leoni S, et al. Patterns of antimicrobial use and antimicrobial resistance among healthy children in Bolivia. Trop Med Int Health 1998;3:11623. [12] Bartoloni A, Bartalesi F, Mantella A, et al. High prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial consumption. J Infect Dis 2004;189:12914. [13] Calva JJ, Sifuentes-Osornio J, Ceron C. Antimicrobial resistance in fecal ora: longitudinal community-based surveillance of children from urban Mexico. Antimicrob Agents Chemother 1996;40:1699702.

312

B. Infante et al. / International Journal of Antimicrobial Agents 25 (2005) 308312 [20] Grape M, Farra A, Kronvall G, Sundstr om L. Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria. Clin Microbiol Infect 2005;11:18592. [21] Grape M, Sundstr om L, Kronvall G. New dfr2 gene as a single gene cassette in a class 1 integron from a trimethoprimresistant Escherichia coli isolate. Microb Drug Resist 2004;9: 31722. [22] Tsao SG, Brunk CF, Pearlman RE. Hybridization of nucleic acids directly in agarose gels. Anal Biochem 1983;131:36572. [23] Lanz R, Kuhnert P, Boerlin P. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet Microbiol 2003;91:7384. [24] Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev 1993;57:13863. [25] Kerrn MB, Klemmensen T, Frimodt-Mller N, Espersen F. Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance. J Antimicrob Chemother 2002;50: 5136.

[14] Lester SC, del Pilar Pla M, Wang F, Perez Schael I, Jiang H, OBrien TF. The carriage of Escherichia coli resistant to antimicrobial agents by healthy children in Boston, in Caracas, Venezuela, and in Qin Pu, China. N Engl J Med 1990;323:2859. [15] Osterblad M, Hakanen A, Manninen R, et al. A between-species comparison of antimicrobial resistance in Enterobacteria in fecal ora. Antimicrob Agents Chemother 2000;44:147984. [16] Olsson-Liljequist B, Larsson P, Walder M, Mi orner H. Antimicrobial susceptibility testing in Sweden. III. Methodology for susceptibility testing. Scand J Infect Dis, Suppl 1997;105:1323. [17] Ringertz S, Olsson-Liljequist B, Kahlmeter G, Kronvall G. Antimicrobial susceptibility testing in Sweden. II. Species-related zone diameter breakpoints to avoid interpretive errors and guard against unrecognized evolution of resistance. Scand J Infect Dis, Suppl 1997;105:812. [18] Kronvall G, Hagelberg A. Numerical evaluation of minimal biochemical test combinations for the identication of Enterobacteriaceae species. APMIS 2002;110:4517. [19] Sambrook KJ, Russel DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001.