Transcript Présentation PowerPoint

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:
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 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
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.
P
5'conserved
segment
attI
cassette 1
intI
attC1
attC1
attI
cassette 2
attC2
attI
attC2
Integron structure
attC1
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.
5’Conserved Segment
P1 P2 attI1
3’ segment
attC
Class 1
intI1
P
Cassette (s)
attI2
qacE∆1 sul1
ORF5
Class 2
intI2
dfrA1 sat aadA1 ORFX
P
tns genes
attI3
Class 3
intI3
Structure of multiresistant integrons (MRI)
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
Cassette
G TTRRRY gene
RYYYAAC---------G TTRRRY
« Inverse Core site »
attC site
Gene-cassette structure
« 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.
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).
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
Integron
IRi
Res
aadA2
intI1
Integron
tetR
sul1
qacED1
floR
orf1 groE/intI1
tet(G)
orf2
pse-1
qacED1/sul1
orf5
IRt
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
IRt
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:
(http://www.ncbi.nlm.nih.gov)
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
RAPD type I and II
dfrA13-oxa5
dfrA7
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+
(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.
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.