International Journal of Antimicrobial Agents 25 (2005) 308312

Acquired sulphonamide resistance genes in faecal Escherichia coli

from 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, 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 first 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 flora 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 affinity for the sulphonamide
inhibitors or, more frequently, from the acquisition of sultype genes that encode DHPSs with reduced affinity 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 first 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 identified 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

cal E. coli of healthy children living in urban areas of Bolivia

and Peru. The finding of the recently described sul3 determinant in this context represents the first 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 significant 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
Identification of E. coli isolates was always confirmed
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 identification procedures [18]. E. coli ATCC 25922 was used as a quality control
organism.

309

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 flanking regions of plasmid pVP440 [1]. PCR amplification 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 amplification 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 specific 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

2.4. Molecular analysis techniques

Basic procedures for DNA extraction, analysis and manipulation were performed as described by Sambrook and
Russel [19]. PCR amplification of sul-type determinants was
carried out with primers designed for specific amplification
of sul1, sul2 and sul3 genes, as described previously [1,9].
Amplification of the entire sul3 gene for sequencing was car-

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

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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

+
+
+
+

Class 1 integrons
sul2

sul3

+
+
+
+
+
+
+

intI1

Variable region (kb)

Gene cassettes

+
+
+
+
+

1.8
1.8
2.8

dfr1, aadA1
dfr1, aadA1
dfr1, catB3, aacA4

1.8

dfr1, aadA1

1.2

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, confirmed 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 profiles. DNA size standard (in kilobases) are shown on the left.

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

sul2 being the most prevalent and sul3 the least common
[1,6,8,9,23]. This is the first 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 specific for the intI1 and
qacE1 genes. In those cases, amplification 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 amplification 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 amplification 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 amplification
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 findings have been reported
earlier and could either reflect 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

311

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.
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