integrons in enteric bacteria isolated in senegal ( subsaharan-africa ) amy gassama sow, pharmad,...
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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
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.
attC1attI
attI
attC2 attC1attI
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
attI1 attC
Cassette (s)
Class 1
Class 2
Class 3
attI3
attI2
intI3
intI1
intI2
qacE∆1 sul1 ORF5
tns genes
P1 P2
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
qacE1 sul1 orf513
catA2 Région 3’
In7dfrA10
In35orf3blaCTX-M-2
orf orf1005blaCTX-M-9
In60
Structure of complex class 1 integrons In6, In7, In35 and In60
Arduino and al., 2002, AAC, 46 (7), 2303-06
Res
IRi
intI1
aadA2
qacE1
sul1
floR
tetR
tet(G)
orf1
orf2
groE/intI1
pse-1
qacE1/sul1
orf5 orf6
IRt
IS6100
IRt
SO44
Salmonella Typhimurium DT104 96-5227 Genomic Island (SGI1)
Integron Integron
Boyd and al. 2000, FEMS, 189, 285-291
Boyd and al. 2001, JCM, 183, 5725-32
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
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+ (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.