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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:

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INTEGRONS IN ENTERIC BACTERIA ISOLATED IN SENEGAL ( SUBSAHARAN-AFRICA )

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Integrons in enteric bacteria isolated in senegal subsaharan africa

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]


Integrons in enteric bacteria isolated in senegal subsaharan africa

Professional experience : TEACHING and RESEARCH

TEACHING:

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

RESEARCH:

Researcher in PASTEUR Institute (Dakar, Senegal):


Integrons in enteric bacteria isolated in senegal subsaharan africa

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


Integrons in enteric bacteria isolated in senegal subsaharan africa

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


Integrons in enteric bacteria isolated in senegal subsaharan africa

  • 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.


Integrons in enteric bacteria isolated in senegal subsaharan africa

P

attI

5'conserved segment

cassette 1

intI

attC1

attI

attC1

cassette 2

attC2

attI

attC2

attC1

Integron structure


Integrons in enteric bacteria isolated in senegal subsaharan africa

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.


Integrons in enteric bacteria isolated in senegal subsaharan africa

5’Conserved Segment

3’ segment

P1

P2

attI1

attC

Class 1

intI1

Cassette (s)

qacE∆1

sul1

ORF5

P

attI2

Class 2

intI2

dfrA1

sat

aadA1

ORFX

tns genes

P

attI3

Class 3

intI3

Structure of multiresistant integrons (MRI)


Integrons in enteric bacteria isolated in senegal subsaharan africa

  • 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


Integrons in enteric bacteria isolated in senegal subsaharan africa

Cassette

RYYYAAC---------G TTRRRY

G TTRRRY gene

« Inverse Core site »

« Core site »

attC site

Gene-cassette structure


Integrons in enteric bacteria isolated in senegal subsaharan africa

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.


Integrons in enteric bacteria isolated in senegal subsaharan africa

  • Super-integrons

  • The super-integrons have been described on the chromosome of different species ofVibrio, 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.


Integrons in enteric bacteria isolated in senegal subsaharan africa

  • 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).


  • Integrons in enteric bacteria isolated in senegal subsaharan africa

    Structure of complex class 1 integrons In6, In7, In35 and In60

    Arduino and al., 2002, AAC, 46 (7), 2303-06

    5’ CS

    cassettes

    qacED1

    sul1

    orf513

    In6

    3’ segment

    catA2

    Région 3’

    In7

    dfrA10

    In35

    blaCTX-M-2

    orf3

    In60

    blaCTX-M-9

    orf

    orf1005


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    Integron

    Integron

    IRt

    IRi

    tetR

    orf1

    groE/intI1

    qacED1/sul1

    IRt

    aadA2

    sul1

    pse-1

    Res

    intI1

    qacED1

    floR

    tet(G)

    orf2

    orf5

    orf6

    SO44

    IS6100

    Salmonella Typhimurium DT104 96-5227 Genomic Island (SGI1)

    Boyd and al. 2000, FEMS, 189, 285-291

    Boyd and al. 2001, JCM, 183, 5725-32


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • Materials and Methods

    • 1- Bacterial strains

    • 10enteroinvasive 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


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • 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 asSHV-12.


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • 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: (http://www.ncbi.nlm.nih.gov)


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • Conjugation experiments

      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.


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • 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


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • 3-Results

    • Mapping of integrons

    • EIEC : class 1 integrons were detected in 4/10

    • 2 RAPD profiles, 1 integron with a single cassettedfrA5

    • 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


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    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


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    • Transfert of antibiotic resistance

    • EIEC

    • All antibiotic resistances expressed by the 4 souches intI1+(AmRSmRSuRTeRTpR) were transferred « en bloc » from each strain to E. coli recipient strain.

    • EaggEC

    • All antibiotic resistances expressed by the 15 souches intI1+(AmRSmRSuRTeRTpR),except resistance to chloramphenicol

    • were transferred « en bloc » from each strain to E. coli recipient strain.


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    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.


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    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


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    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.


    Integrons in enteric bacteria isolated in senegal subsaharan africa

    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.


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