INTEGRONS IN ENTERIC BACTERIA ISOLATED IN SENEGAL ( SUBSAHARAN-AFRICA ) Amy GASSAMA SOW, PharmaD, PhD Laboratoire de Bactériologie Expérimentale, Institut Pasteur, 220, Dakar, Sénégal

Professional experience : TEACHING and RESEARCH TEACHING: Assistant Professor in the Department of Applied Biology and Chemical Engineering, Ecole Supérieure de Polytechnique, Université C. A. DIOP, Dakar, Senegal: Full-time pratical and theoretical teaching in Medical Parasitology and Microbiology RESEARCH: Researcher in PASTEUR Institute (Dakar, Senegal):

Aetiologies, and pathogenesis of bacterial gastroenteritis: -Phenotypic and genotypic characterization of enteric pathogens isolated in human and food. -Research on aetiologies of diarrheas in immunocompetent and immunocompromised patients. -Identification of a new ribotype of Vibrio cholerae during the last Senegalese cholera outbreak in Molecular mechanisms of antibiotics resistance, molecular mechanisms of transmission of resistance genes: -Characterization of integrons in enteric bacteria -Transfer of antibiotics resistance genes in enteric bacteria

Introduction Resistance to antibiotics is increasing in enteropathogenic bacteria In Africa: high rate of resistance to ampicillin, tetracyclin, sulfonamides and trimethoprim Few data are available on the mechanisms of resistance in diarrhegenic bacteria

Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like : Plasmids Transposons Integrons are a system of gene capture and expression composed of an intI gene encoding an integrase, a recombination site attI, and a promoter.

attC1 attI attC2attC1 attI attC1 attC2 5'conserved segment cassette 1 cassette 2 Integron structure intI P

Several classes of integrons have been described according to the sequence of intI gene. Three (class 1, 2, 3) of them are well characterized and are involved in antibiotic resistance The integrase is able to integrate or excise gene cassettes, by a site-specific system of recombination. Cassette mobility results in a very efficient system of dissemination of resistance genes.

Structure of multiresistant integrons (MRI) 5’Conserved Segment 3’ segment attI1attC Cassette (s) Class 1 Class 2 Class 3 attI3 attI2 intI3 intI1 intI2 qacE∆1sul1ORF5 tns genes P1P2 P P ORFXaadA1satdfrA1

Cassettes exhibit variable size (260 à 1500 bp) but have common strucure Cassettes generally consist of a single gene and a short sequence located downtream of the gene that is a site specific reombination, called « 59-bas element », attC site Over 100 cassettes have been described at present count Cassettes can exist as free circular DNA molecules, but normally found integrated in a linearized form in an integron

G TTRRRY gene RYYYAAC G TTRRRY « Inverse Core site » attC site « Core site » Gene-cassette structure Cassette

The attC site is bounded by the core site and the inverse core site. The cross-over point occurs between the G base of a core site GTTRRRY and the first T base of a second core site. The gene cassettes in an integron are expressed from a common promotor region located in the 5’CS of the integron. The level of the expression of cassette-associated genes may be affected by their position within the integron.

Super-integrons The super-integrons have been described on the chromosome of different species of Vibrio, Pseudomonas, Shewanella, Xanthomonas, Listonnella, Nitrosomonas. SI contain:  intI gene  site specific attI  Repeat sequences separated by open reading frame The super-integrons could constitute the source of the three classes of integrons involved in the dissemination of antibiotic resistance.

Complex class 1 integrons Described on plasmids pSa et pDGO100 : In6 et In7 These integrons contain a partial duplication of the 3’segment and carry antibiotic resistance genes. Between the two 3’ segments, there is a region which includes ORF 513 (Genbank accession number L06418).

In6 5’ CS cassettes 3’ segment qacE  1 sul1orf513 catA2Région 3’ In7 dfrA10 In35 orf3 bla CTX-M-2 orf orf1005 bla CTX-M-9 In60 Structure of complex class 1 integrons In6, In7, In35 and In60 Arduino and al., 2002, AAC, 46 (7),

Res IRi intI1 aadA2 qacE  1 sul1 floR tetR tet(G) orf1 orf2 groE/intI1 pse-1 qacE  1/sul1 orf5orf6 IRt IS6100 IRt SO44 Salmonella Typhimurium DT Genomic Island (SGI1) Integron Boyd and al. 2000, FEMS, 189, Boyd and al. 2001, JCM, 183,

Materials and Methods 1- Bacterial strains  10 enteroinvasive E. coli (EIEC) et 25 enteroaggregative E. coli (EAggEC) isolaed from diarrheal adult patients in Dakar. Strains were resistant to trimethoprim, sulfonamides, ampicillin, tetracyclines, chloramphenicol, streptomycin and spectinomycin

 08 strains of Salmonella enterica serovar Keurmassar, isolated from human (7 strains from stools and blood) and poultry (1 strain from flesh) Strains were resistant to ampicillin, chloramphenicol, sulfonamides, tetracyclines, tobramycin, gentamycin, streptomycin, spectinomycin and trimethoprim. The eight strains expressed an extended-spectrum betalactamase which was identified as SHV-12.

2- Methods  PCR mapping Strains were screened for the presence of class 1, 2, and 3 integrons by PCR using three sets of primers specific for intI1, intI2, intI3 genes Cassettes assortment in class 1 integrons was determined by amplification with primers annealing to the 5’ and 3’ ends. PCR products were sequenced directly by using the ABI PRISM dRhodamine protocole Nucleotide sequence analysis was obtained at the National Center of Biotechnology Information Website: (

 Conjugation experiments Transfer of antibiotic resistance from EIEC, EaggEC, Salmonella Keumassar strains to E. coli recipient strain resistant to nalidixic acid was achieved on a selective medium containing 50µg/ml, either 5 µg/ml of trimethoprim or 25 µg/ml of streptomycin Tests for the presence and content of integrons were done as described above. Plasmid were extracted by alkaline lysis method.

 Molecular typing The molecular typing was done by Random Amplified polymorphism DNA (RAPD) for EIEC and EaggEC. Pulsed field gel electrophoresis (PFGE) was done for Salmonella Keurmassar

3-Results  Mapping of integrons EIEC : class 1 integrons were detected in 4/10 2 RAPD profiles, 1 integron with a single cassette dfrA5 EAggEC : class 1 integrons were detected in 15/25 4 profils RAPD 3 class 1 integrons: aadA1 dfrA13-oxa5 dfrA7 RAPD type I and II RAPD type III

Salmonella Keurmassar : class 1 integrons were detected in all strains 1 PFGE patterns, 2 class 1 integrons aadA2 aac(6’)-IIc-ereA2 aadA confer resistance to streptomycin and spectinomycin aac(6’)-IIc confers resistance to gntamicin, netilmicin and tobramycin ereA2 encodes resistance to erythromycin Neither class 2 nor class 3 integrons were detected

 Transfert of antibiotic resistance EIEC All antibiotic resistances expressed by the 4 souches intI1+ (Am R Sm R Su R Te R Tp R ) were transferred « en bloc » from each strain to E. coli recipient strain. EaggEC All antibiotic resistances expressed by the 15 souches intI1+ (Am R Sm R Su R Te R Tp R ), except resistance to chloramphenicol were transferred « en bloc » from each strain to E. coli recipient strain.

The PCR analysis of all transconjugants confirmed the transfer of class 1 integrons Salmonella Keurmassar All antimicrobial drug resistances were transferred at once from each strain to E. coli resistant to nalidixic acid. The analysis of plasmid from all transconjugants showed a single plasmid of > 30Kb. The PCR analysis confirmed the transfer of two integrons which suggested that the integrons were borne by a conjugative plasmid.

Conclusion Integrons detected in EIEC and in EaggEC are determinant for trimethoprim resistance (dfrA5, dfrA7, dfrA13), or spectinomycin or streptomycin resistance (aadA1). Trimethoprim in combination to sulfamethoxazole is the first line drug used to treat diarrheal illnesses in Senegal Streptomycin was use in diarrheal diseases and tuberculosis Spectinomycin was used to treat gonococi. The selective pressure has lead to the emergence and dissemination of strains harboring such integrons

In Salmonella Keurmassar, determining how the cassette combination aac (6’)-IIc-ereA2 was selected is difficult. This zoonotic species could acquire its cassette in poultry, but investigation has failed to prove any relationship between the animal and human isolates. Aminoglycosides are not used extensively in Senegal because they are expensive. However, erythromycin is extensively use in poultry industry to reduce deaths and increase productivity. The finding of a single plasmid >30Kb, harboring resistance determinants to streptomycin, spectinomycin, and erythromycin resistance genes raises the possibility that the use of these antibiotics could co-selected aac (6’)-IIc cassette.

Furthermore, in Senegal, antibiotics are sold over the counter, which leads to self-medication thus increasing selective pressure. Our findings showed that the horizontal transfer of integrons plays a dominant role in the development of multiresistance in enteric pathogens. Active surveillance of antimicrobial use in animal husbandry and human medecine is important to reduce selective pressure and subsequent dissemination of mutiresistant strains.