Denis 2,3,4, and Marie-Cécile Ploy 2,3,4

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.


Introduction
Shigellosis, which is primarily a disease of resource-poor populations, is an important cause of morbidity and mortality among people of all ages living in communities lacking adequate sanitation and safe water. Children under five years old in developing countries are particularly at risk. An estimated 160 million cases and 1.1 million deaths per year are due to shigellosis [1]. Shigella flexneri is the most prevalent serotype in Africa [2][3][4]. Shigellae are noteworthy for their multiple drug resistance, having gradually acquired resistance to most widely used and inexpensive antimicrobial drugs [5][6][7][8].
Mobile genetic elements such as plasmids and transposons are involved in the spread of resistance, together with genomic islands and integrons. Integrons are genetic elements that, by site-specific recombination, can integrate gene cassettes, usually antibiotic resistance genes [9]. Class 1 and 2 are the most frequent in Gram-negative bacteria [10]. The structure of class 1 integron includes 5' and 3' conserved segments and a variable region containing gene cassettes. Most previously described class 2 integrons contain the same array of four gene cassettes, three antibiotic resistance gene cassettes (dfrA1, sat and aadA1), conferring resistance to trimethoprim, streptothricin, and spectinomycin/streptomycin, respectively), and the orfX cassette of unknown function. Class 2 integrons have been described in transposon Tn7 and its derivatives, and their 3' segment contains five tns genes involved in transposon movements [11]. The movement of cassettes are catalyzed by site-specific recombination; cassette mobility results in the dissemination of resistance genes.
Integrons have a well-established role in the dissemination of resistance among Gram-negative pathogens and are thus a useful marker of antibiotic resistance [12]. Trimethoprim is widely used to treat several infectious diseases in Senegal, in combination with sulfonamides. As trimethoprim resistance determinants are often found in gene cassettes, we examined the prevalence of integrons in Senegalese Shigella isolates.

Bacterial isolates
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 hospital and an urban general hospital in Dakar, Senegal, were included in the study.

Antimicrobial susceptibility
Antibiotic susceptibility was tested with the disk diffusion method on Mueller-Hinton agar (Becton Dickinson Cokeysville, Md), and antibiotics were those used for Enterobacteriaceae, as recommended by the Clinical Laboratories Standards Institute (www.clsi.org).

DNA sequencing
Purified PCR products were sequenced on an 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 resistance
All the strains were resistant to at least four of the following antibiotics: ampicillin, ticarcillin, tetracycline, trimethoprim, sulfamethoxazole, streptomycin, and chloramphenicol (Table 1).

Int genes
Twenty-eight (87.5%) of the 32 strains contained at least one integron, and 26 of these 28 strains contained at least one class 1 integron. The intI2 gene was detected in 16 strains, either alone (2 strains of S. flexneri) or together with intI1 (14 strains). The intI1 and intI2 genes were found together in all S. boydii 20 isolates, and in 9 of the 14 S. dysenteriae isolates. No class 3 integrons were detected.

Cassette arrays
In 3' segment-containing class 1 integrons, the gene cassettes arrays were characterized by PCR with primers 5'CS and 3'CS and by sequencing the amplification products. The strains yielded amplicons ranging from 0.7 kb to 1.8 kb. Four different cassette arrays with one (dfrA5, dfrA15) or 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 cassettes, of which aadA1 was most prevalent. To determine the cassette arrays of these strains, amplification was performed with primers 5'CS 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. described Shigella strains containing a class 1 integron in which these two cassettes were followed by the insertion sequence IS1. Successful amplification with primers AadA3 and is1b (5'-GTGAACGCACTATGGCGACGC TG-3'), located within IS1 [17], confirmed that the genetic environment was the same as that described by Dubois et al. [18].
The class 2 integrons of our strains are heterogenous; two S. flexneri strains harbouring exclusively class 2 integrons had a classical organization with the same four cassettes as those found in Tn7, dfrA1, sat, aadA1 and orfX. Either The aadA1 or the orfX cassette was lacking in all strains containing both class 1 and 2 integrons ( Table 1).

Transfer of antibiotic resistance and genetic location of integrons
Resistance to ampicilin, ticarcillin, streptomycin, tetracycline, sulfamethoxazole, trimethoprim, excepted chloramphenicol for one strain of Shigella dysenteriae was transferred simultaneously from all Shigella strains harbouring class 1 integrons with cassette arrays (dfrA15, dfrA15-aadA1, dfrA1-aadA2). Conjugation experiments were unsuccessful for strains harbouring unusual class 1 and 2 integrons supporting the chromosomal location of these integrons. Strains harbouring the classical class 1 and 2 integrons yielded transconjugants on trimethoprim suggesting their plasmidic location.

Distribution of integrons among RAPD types and serotypes
Six RAPD profiles were identified, three in Shigella dysenteriae (A, B, C), two in Shigella flexneri (D, E), and one in Shigella boydii 20 (F) (  belonging to the B profile did not contain integrons. All Shigella dysenteriae and the majority of Shigella flexneri strains (11/12) harbouring integrons showed a unique RAPD profile but had a different integron carriage except Shigella boydii 20 strains which showed a unique profile with the same integron content (Table 1). These data revealed a clonal spread of integrons among serotypes and a transfer of integrons between different serotypes.

Discussion
Antibiotic resistance is common in Shigella spp. [7,19,4,20]. In Senegal, multiple resistance to ampicillin, sulfamethoxazole, trimethoprim and tetracycline is related to the intensive first-line use of these antibiotics to treat diarrhoeal illnesses and other infectious diseases. Here we found that 87.5% of 32 Shigella strains isolated in Senegal contained at least one integron. This marked dissemination of integrons among Shigella spp is partly linked to the propensity of this genus to acquire plasmids, as multidrug resistance integrons are usually plasmidborne [18]. In this study, class 1 integrons were the most prevalent, in keeping with the results of other studies of African Shigella isolates [18]. In contrast, integron prevalence is very low in other parts of the world [20,21,22]. We found class 2 integrons in half the 32 strains studied here. Other studies have shown that class 2 integrons are more prevalent in industrialized and/or emerging countries [23,24,25]. Among shigellae, class 2 integrons tend to be associated with Shigella sonnei [26,20,27,28]. We detected class 1 and 2 integrons in S. dysenteriae, S. flexneri and in the new serovar of S. boydii 20. The latter was first isolated in Canada from patients who had recently travelled to Cuba, Ethiopia, India, Guatemala or Mexico [28]. In Senegal, this serovar was identified between July and September 2000.
Class 1 integrons were highly diverse, with five different integrons harbouring a dfr cassette, alone or associated with aadA1. Furthermore, all class 2 integrons detected contained the dfrA1 cassette and 8 out of 16 also contained aadA1 cassette. Our results thus showed that trimethoprim and streptomycin/spectimonycin resistant gene cassettes were highly encountered in Senegalese Shigella isolates. In sub-Saharan Africa, trimethoprim is widely used, in combination with sulfonamides, to treat diarrhoeal illnesses and urinary tract infections as well as to prevent opportunistic infections in HIV-infected and malarial patients [29,30]. Streptomycin is intensively used to treat tuberculosis, in combination with other drugs. The high prevalence of dfr and aadA1 gene cassettes in integron-containing Shigella strains may thus be related to the use of these antibiotics. However, the dfr and aadA cassettes are also very common in integrons harboured by other Enterobacteriaceae species isolated from patients in industrialized countries [20,21]. In a previous study in Senegal we also found a high prevalence of dfr and aadA cassettes in integron-containing enteroaggregative and enteroinvasive Escherichia coli (E. coli) strains [31]. Horizontal transfer could readily occur between E. coli and Shigella, which are both enteric pathogens. Indeed, we successfully obtained transconjugants with dfr-containing strains. These two studies showing a high prevalence of dfrcontaining integrons in enteric pathogens strongly challenge the use of trimethoprim in Senegal.
We detected the unusual class 1 integron with partial or total deletion of the 3' segment, as previously described in Shigella dysenteriae and Shigella flexneri [26,18]. In our strains this atypical class 1 integron was found either alone or associated with a class 2 integron lacking the aadA1 or orfX cassette. This type of integron was associated to multiple resistance (as shown in Table  1) and confirm the role of integron in antibioticresistance. Otherwise, the deletion of the aadA1 cassette could result from intI1 integrase-catalyse co-integrate formation between a class 1 and 2 integron or a possible RecA-dependent homologous recombination between two copies of the same cassette in both classes of integron [32].
Integron carriage was unrelated to the RAPD profile in Shigella dysenteriae and Shigella flexneri, whereas Shigella boydii 20 strains had a unique profile with the same integron content, indicating clonal spread. Previous studies also found similar patterns for S. boydii serotype 20 by using pulsedfield gel electrophoresis and ribotyping, and inferred that this serotype could be homogeneous [28,33].
The detection of integrons in a new Shigella serovar, in addition with high integron prevalence among Shigella strains, confirms the propensity of shigellae to acquire and disseminate resistance determinants.
The presence of integrons in Shigella may have important clinical implications, as multiple gene cassettes could be captured by such strains easily leading to multidrug resistance, even to broadspectrum antibiotics such as third-generation cephalosporins and quinolones.