INTEGRONS IN ENTERIC BACTERIA ISOLATED IN SENEGAL ( SUBSAHARAN-AFRICA ). Amy GASSAMA SOW, PharmaD, PhD Laboratoire de Bactériologie Expérimentale, Institut Pasteur, 220, Dakar, Sénégal [email protected] Professional experience : TEACHING and RESEARCH TEACHING:
ENTERIC BACTERIA ISOLATED
Amy GASSAMA SOW, PharmaD, PhD
Laboratoire de Bactériologie Expérimentale, Institut Pasteur, 220, Dakar, Sénégal
Professional experience : TEACHING and RESEARCH
Assistant Professor in theDepartment of Applied Biology and Chemical Engineering, Ecole Supérieure de Polytechnique, Université C. A. DIOP, Dakar, Senegal:
Full-time pratical and theoretical teaching in MedicalParasitology and Microbiology
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 1994.
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
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
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)
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
« Inverse Core site »
« Core site »
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.
The super-integrons could constitute the source of the three classes of integrons involved in the dissemination of antibiotic resistance.
Structure of complex class 1 integrons In6, In7, In35 and In60
Arduino and al., 2002, AAC, 46 (7), 2303-06
Salmonella Typhimurium DT104 96-5227 Genomic Island (SGI1)
Boyd and al. 2000, FEMS, 189, 285-291
Boyd and al. 2001, JCM, 183, 5725-32
Strains were resistant to trimethoprim, sulfonamides, ampicillin, tetracyclines, chloramphenicol, streptomycin and spectinomycin
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
Transferof 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 5µg/ml of trimethoprim or 25µg/ml of streptomycin
Tests for the presence and content of integrons were done as described above. Plasmid were extracted by alkaline lysis method.
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
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
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
The PCR analysis of all transconjugants confirmed the transfer of class 1 integrons
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.
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-ereA2was 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.