High prevalence of trimethoprim-resistance cassettes in class 1 and 2 integrons in senegalese shigella spp isolates

Original Article

High prevalence of trimethoprim-resistance cassettes in class 1 and 2
integrons in Senegalese Shigella spp isolates

Amy Gassama-Sow1, Awa Aïdara-Kane1§, Olivier Barraud2,3,4, Martine Gatet2,3, François
Denis2,3,4, and Marie-Cécile Ploy2,3,4
1Unité de Bactériologie Expérimentale, Institut Pasteur, 220, Dakar, Sénégal 2Université de Limoges, Faculté de Médecine, EA 3175, Limoges, 87000, France 3INSERM, Equipe Avenir, Limoges 87000, Limoges, France 4 CHU Limoges, Laboratoire de Bactériologie-Virologie-Hygiène, 87000, Limoges, France § Present address: World Health Organization, Food Safety and Zoonoses (FOS), Health Security and Environment (HSE),
20 Avenue Appia, CH-1211 Geneva 27, Switzerland

Abstract
Background: Integrons have a well-established role in the dissemination of resistance among Gram-negative pathogens and are thus a
useful marker of antibiotic resistance. Shigellae are noteworthy for their multiple drug resistance, having gradually acquired resistance to
most widely use and inexpensive antimicrobial drugs.
Methodology: A total of 32 Shigella strains belonging to serotypes flexneri, dysenteriae, and boydii 20, a new Shigella serovar, resistant
to at least four antibiotics were analyzed by molecular techniques.
Results: Class 1 integrons were the most prevalent (92.8%); class 2 integrons were found in 16 strains (57.1%). Fifty percent of the
strains harboured both class 1 and 2 integrons (intI1 and intI2 genes); this combination of integrase genes was most prevalent in S. boydii
20 and S. dysenteriae strains. The class 1 integrons detected contained dfr and aadA cassettes, alone or in combination (dfrA5/dfrA15, or
dfrA15-aadA1, dfrA1-aadA2), and an atypical cassette array with an insertion sequence (oxa30-aadA1-IS1). For class 2 integrons, we
detected either the same cassettes as those found in Tn7 (dfrA1-sat1-aadA1-orfX) or truncated class 2 integrons without aadA1 or orfX.
The tns genes were absent from all class 2 integrons.
The distribution of integrons among RAPD profiles and serotypes revealed a clonal spread of integrons into serotypes and a transfer of
integrons between different serotypes.
Conclusions: The detection of integrons in a new Shigella serovar, in addition with a high integron prevalence among Shigella strains,
confirms the propensity of shigellae to acquire and disseminate resistance determinants.

Key words: multi-resistant Shigella; integrons; dfr

J Infect Dev Ctries 2010; 4(4):207-212.

(Received 15 October 2009 - Accepted 17 February 2010)
Copyright 2010 Gassama-Sow et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
antibiotic resistance genes [9]. Class 1 and 2 are the Shigellosis, which is primarily a disease of most frequent in Gram-negative bacteria [10]. The resource-poor populations, is an important cause of structure of class 1 integron includes 5’ and 3’ morbidity and mortality among people of all ages conserved segments and a variable region living in communities lacking adequate sanitation and safe water. Children under five years old in described class 2 integrons contain the same array developing countries are particularly at risk. An of four gene cassettes, three antibiotic resistance estimated 160 million cases and 1.1 million deaths gene cassettes (dfrA1, sat and aadA1), conferring per year are due to shigellosis [1]. Shigella flexneri resistance to trimethoprim, streptothricin, and is the most prevalent serotype in Africa [2-4]. spectinomycin/streptomycin, respectively), and the Shigellae are noteworthy for their multiple drug orfX cassette of unknown function. Class 2 resistance, having gradually acquired resistance to integrons have been described in transposon Tn7 most widely used and inexpensive antimicrobial and its derivatives, and their 3’ segment contains five tns genes involved in transposon movements Mobile genetic elements such as plasmids and [11]. The movement of cassettes are catalyzed by transposons are involved in the spread of resistance, together with genomic islands and integrons. results in the dissemination of resistance genes. Integrons are genetic elements that, by site-specific Integrons have a well-established role in recombination, can integrate gene cassettes, usually the dissemination of resistance among Gram- Gassama-Sow et al. - Trimethoprim-resistance in Shigella spp isolates J Infect Dev Ctries 2010; 4(4):207-212. negative pathogens and are thus a useful marker of antibiotic resistance [12]. Trimethoprim is widely 3’), aadA3 (5’-GAATGATGTCGTGCACAAC-3’) used to treat several infectious diseases in Senegal, located in the aadA1 gene cassette (this study), and in combination with sulfonamides. As trimethoprim resistance determinants are often found in gene cassettes, we examined the prevalence of integrons To characterize the 3’ segment of class 2 integrons, amplification was performed using Materials and methods
TACCTGTTCTGCCCGTATCT-3’) and int7S (5’- A total of thirty-two Shigella strains belonging to serotypes flexneri (N = 14), dysenteriae (N = 13), and Shigella boydii 20 serovar nov. (N = 5) isolated from adults with diarrhoea at a teaching Purified PCR products were sequenced on an hospital and an urban general hospital in Dakar, ABI Prism automatic sequencer, as recommended by the manufacturers; the nucleotide sequences were compared online at the National Center for Biotechnology Information (NCBI) website. Antibiotic susceptibility was tested with the disk diffusion method on Mueller-Hinton agar Mating experiments were conducted in Luria- antibiotics were those used for Enterobacteriaceae, Bertani broth with the nalidixic acid-resistant E. as recommended by the Clinical Laboratories coli strain C1a as recipient. Transconjugants were selected on Luria-Bertani agar plates containing nalidixic acid (50 µg/ml), and trimethoprim (100 Total DNA was extracted with the Qiamp DNA transconjugants were screened for intI1 and intI2 by minikit (Qiagen, S.A, Courtaboeuf, France). Random amplified polymorphic DNA analysis The strains were screened by PCR for class 1, 2 and 3 integrons with the primers described above [13,14,15]. The 50-µl PCR reaction mix consisted
of Taq polymerase buffer, 1.5 mM MgCl2, 200 µM [15] (Bioprobe Systems, Montreuil sous bois, deoxynucleotide triphosphates, 50 pmol of each France). Strains were considered non-identical if primer (Isoprim, Toulouse, France), 1 U of Taq, their RAPD patterns differed by at least two bands. and 25 ng of DNA were amplified in a thermal cycler (Perkin-Elmer 2400, Applied Biosystems). All the strains were resistant to at least four of sul1 (5’-GTCCGACATCCACGACGTCTGATC- the following antibiotics: ampicillin, ticarcillin, 3’) were used to detect the 3’ segment usually found in class 1 integrons [15]. PCR amplification streptomycin, and chloramphenicol (Table 1). of the class 1 integron cassette array used primers 5’CS (5’-GGCATCCAAGCAGCAAG-3’) and 3’CS (5’-AAAGCAGACTTGACCTGA-3’) [15]. If the 3’ segment was absent, amplification was contained at least one integron, and 26 of these 28 performed with primers located in cassettes strains contained at least one class 1 integron. The selected on the basis of the resistance phenotypes. intI2 gene was detected in 16 strains, either alone (2 When a strain yielded two PCR products, the strains of S. flexneri) or together with intI1 (14 strains). The intI1 and intI2 genes were found electrophoresis and purified with the QIAquick gel together in all S. boydii 20 isolates, and in 9 of the extraction kit (Qiagen, S. A, Courtaboeuf, France). 14 S. dysenteriae isolates. No class 3 integrons The cassette content of class 2 integrons was characterized with primers located in the cassettes usually found in class 2 integrons: hep74 (5’- Gassama-Sow et al. - Trimethoprim-resistance in Shigella spp isolates J Infect Dev Ctries 2010; 4(4):207-212. Table 1. Characteristics of Shigella strains: Resistance phenotypes, cassette arrays, and RAPD profiles
oxa30-aadA1-IS1/6 (E), 1 (D) * class 1 integrons
** class 2 integrons
Amp, ampicillin; Tic, ticarcillin; Te, tetracycline; Str, streptomycin; SSS, sulfamethoxazole, Tmp, trimethoprim; Cm, chloramphenicol In 3’ segment-containing class 1 integrons, the organization with the same four cassettes as those gene cassettes arrays were characterized by PCR found in Tn7, dfrA1, sat, aadA1 and orfX. Either
with primers 5’CS and 3’CS and by sequencing the The aadA1 or the orfX cassette was lacking in all strains containing both class 1 and 2 integrons amplicons ranging from 0.7 kb to 1.8 kb. Four different cassette arrays with one (dfrA5, dfrA15) or Transfer of antibiotic resistance and genetic two cassettes (dfrA15-aadA1, dfrA1-aadA2) were characterized (Table 1). The 14 strains lacking the 3’ segment were resistant to streptomycin and spectinomycin, owing to the presence of aadA gene trimethoprim, excepted chloramphenicol for one cassettes, of which aadA1 was most prevalent. To strain of Shigella dysenteriae was transferred determine the cassette arrays of these strains, simultaneously from all Shigella strains harbouring amplification was performed with primers 5’CS class 1 integrons with cassette arrays (dfrA15, and aadA3 yielding a PCR product of 1.5 kb. Sequencing of this product showed the presence of two cassettes, oxa30 and aadA1. Dubois et al. harbouring unusual class 1 and 2 integrons described Shigella strains containing a class 1 supporting the chromosomal location of these integron in which these two cassettes were followed integrons. Strains harbouring the classical class 1 by the insertion sequence IS1. Successful and 2 integrons yielded transconjugants on amplification with primers AadA3 and is1b (5’- trimethoprim suggesting their plasmidic location. located within IS1 [17], confirmed that the genetic Distribution of integrons among RAPD types and environment was the same as that described by Six RAPD profiles were identified, three in The class 2 integrons of our strains are Shigella dysenteriae (A, B, C), two in Shigella heterogenous; two S. flexneri strains harbouring flexneri (D, E), and one in Shigella boydii 20 (F) exclusively class 2 integrons had a classical (Table 1). Two Shigella dysenteriae strains Gassama-Sow et al. - Trimethoprim-resistance in Shigella spp isolates J Infect Dev Ctries 2010; 4(4):207-212. belonging to the B profile did not contain integrons. related to the use of these antibiotics. However, the All Shigella dysenteriae and the majority of dfr and aadA cassettes are also very common in Shigella flexneri strains (11/12) harbouring integrons harboured by other Enterobacteriaceae integrons showed a unique RAPD profile but had a species isolated from patients in industrialized different integron carriage except Shigella boydii countries [20,21]. In a previous study in Senegal we 20 strains which showed a unique profile with the also found a high prevalence of dfr and aadA same integron content (Table 1). These data cassettes in integron-containing enteroaggregative revealed a clonal spread of integrons among and enteroinvasive Escherichia coli (E. coli) strains serotypes and a transfer of integrons between [31]. Horizontal transfer could readily occur between E. coli and Shigella, which are both enteric pathogens. Indeed, we successfully obtained Discussion
transconjugants with dfr-containing strains. These Antibiotic resistance is common in Shigella two studies showing a high prevalence of dfr- spp. [7,19,4,20]. In Senegal, multiple resistance to containing integrons in enteric pathogens strongly ampicillin, sulfamethoxazole, trimethoprim and challenge the use of trimethoprim in Senegal. tetracycline is related to the intensive first-line use We detected the unusual class 1 integron with of these antibiotics to treat diarrhoeal illnesses and partial or total deletion of the 3’ segment, as other infectious diseases. Here we found that 87.5% previously described in Shigella dysenteriae and of 32 Shigella strains isolated in Senegal contained Shigella flexneri [26,18]. In our strains this atypical at least one integron. This marked dissemination of class 1 integron was found either alone or integrons among Shigella spp is partly linked to the associated with a class 2 integron lacking the propensity of this genus to acquire plasmids, as aadA1 or orfX cassette. This type of integron was multidrug resistance integrons are usually plasmid- associated to multiple resistance (as shown in Table borne [18]. In this study, class 1 integrons were the 1) and confirm the role of integron in antibiotic- most prevalent, in keeping with the results of other resistance. Otherwise, the deletion of the aadA1 studies of African Shigella isolates [18]. In contrast, cassette could result from intI1 integrase-catalyse integron prevalence is very low in other parts of the co-integrate formation between a class 1 and 2 world [20,21,22]. We found class 2 integrons in integron or a possible RecA-dependent homologous half the 32 strains studied here. Other studies have recombination between two copies of the same shown that class 2 integrons are more prevalent in cassette in both classes of integron [32]. industrialized and/or emerging countries [23,24,25]. Integron carriage was unrelated to the RAPD Among shigellae, class 2 integrons tend to be profile in Shigella dysenteriae and Shigella flexneri, associated with Shigella sonnei [26,20,27,28]. We whereas Shigella boydii 20 strains had a unique detected class 1 and 2 integrons in S. dysenteriae, S. profile with the same integron content, indicating flexneri and in the new serovar of S. boydii 20. The clonal spread. Previous studies also found similar latter was first isolated in Canada from patients patterns for S. boydii serotype 20 by using pulsed- who had recently travelled to Cuba, Ethiopia, India, field gel electrophoresis and ribotyping, and Guatemala or Mexico [28]. In Senegal, this serovar inferred that this serotype could be homogeneous was identified between July and September 2000. Class 1 integrons were highly diverse, with five The detection of integrons in a new Shigella different integrons harbouring a dfr cassette, alone serovar, in addition with high integron prevalence or associated with aadA1. Furthermore, all class 2 among Shigella strains, confirms the propensity of integrons detected contained the dfrA1 cassette and shigellae to acquire and disseminate resistance 8 out of 16 also contained aadA1 cassette. Our results thus showed that trimethoprim and The presence of integrons in Shigella may have streptomycin/spectimonycin resistant gene cassettes important clinical implications, as multiple gene were highly encountered in Senegalese Shigella cassettes could be captured by such strains easily isolates. In sub-Saharan Africa, trimethoprim is leading to multidrug resistance, even to broad- widely used, in combination with sulfonamides, to spectrum antibiotics such as third-generation treat diarrhoeal illnesses and urinary tract infections as well as to prevent opportunistic infections in HIV-infected and malarial patients [29,30]. Acknowledgements
This work was supported by grants from the French tuberculosis, in combination with other drugs. The Ministère de la Recherche, from Conseil Régional du Limousin, and from EGIDE, the French Ministère des Affaires high prevalence of dfr and aadA1 gene cassettes in integron-containing Shigella strains may thus be Gassama-Sow et al. - Trimethoprim-resistance in Shigella spp isolates J Infect Dev Ctries 2010; 4(4):207-212. References
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