BACKGROUND
extended-spectrum β-lactamase -producing bacteria represent a major worldwide threat among drug-resistant bacteria in both hospital and community settings
<cit> .
esbls are among the ambler classes a, confer resistance to β-lactam antibiotics except cephamycins and carbapenems, and are inhibited by clavulanic acid
<cit> .
esbls are often located on large plasmids that also harbor resistant genes to other antimicrobial classes with resulting multidrug-resistant isolates
<cit> .
the first esbls have evolved by genetic mutation from native β-lactamases tem and shv
<cit>  <cit> .
recently, a novel type of esbls, the ctx-m enzymes, emerged worldwide, mostly from enterobacteriaceae <cit> .
ctx-m β-lactamases are not closely related to tem or shv esbls but share high amino-acid identity with chromosomal β-lactamases from kluyvera spp.
<cit> .
now, blactx-m- <dig> is recognized as the most widely distributed ctx-m enzyme
<cit> .
it is derived from ctx-m- <dig> by a substitution of asp-240-gly which increases its catalytic efficiency against ceftazidime
<cit> .
blactx-m- <dig> are encoded on plasmids belonging to the incompatibility group incf
<cit> .
in the upstream region of ctx-m genes an insertion sequence element, isecp <dig>  is commonly present and is likely responsible for the transposition process of the genes
<cit> .
e. coli is among the most prevalent causes of hospital-acquired and community-acquired bacterial infections and their resistances to antimicrobial agents have become a serious concern for healthcare providers
<cit> .
phylogenetic analyses have classified e. coli into four main phylogenetic groups .
commensal isolates belong mainly to a and b <dig> groups whereas virulent extra-intestinal pathogenic e. coli  are essentially from the b <dig> and d groups
<cit> .
expec harbor numerous virulence factors including α-hemolysin, cytotoxic necrotizing factor, adhesins and iron acquisition systems
<cit> .
the spread of blactx-m- <dig> has been mainly associated with the dissemination of a particular clone of e. coli st <dig> belonging to phylogenetic group b2
<cit> .
recently, an e. coli clone o <dig> st <dig>  producing ctx-m- <dig>  with high virulence potential and belonging to the b <dig> group, has been reported and represent a major public health problem
<cit> .
many reports have documented the emergence of esbl-producing enterobacteriaceae <cit> .
in antananarivo, esbls were first detected in  <dig> from uti in  <dig> % of isolated enterobacteriaceae <cit> .
in  <dig>  outbreaks of ctx-m- <dig> and shv-2-producing k. pneumoniae isolates have been described in two pediatric units
<cit> .
more recently,  <dig> % of clinical isolates from patients in surgery and intensive care units
<cit>  and  <dig> % of intestinal carriage isolates from children hospitalized in a pediatric department of a large teaching hospital
<cit>  were esbl-producers.
for  <dig> multidrug-resistant enterobacteriaceae isolates from antananarivo, we characterized: i) the genes encoding the esbls; ii) the drug resistance genes associated with the esbl genes; iii) gene cassettes present in the isolates; and iv) the plasmid incompatibility groups of the isolates.
we also determined the phylogenetic groups and virulence factors of the e. coli isolates.
methods
ethical clearance
the study protocols were approved by the national ethics committee of madagascar.
written informed consents were obtained from all patients and at least one parent of each child before enrollment.
patients
between september  <dig> and december  <dig>  a total of  <dig> non-duplicate bacterial isolates were obtained from  <dig> patients.
<dig> patients were recruited from several wards in four hospitals in antananarivo, madagascar  and  <dig> patients referred to the pasteur institute medical laboratory in antananarivo.
laboratory methods
various clinical specimens  were collected and submitted for bacterial analysis at the pasteur institute medical laboratory in antananarivo.
presumptive enterobacteria isolates were identified using standard microbiological methods and the api 20e system .
antimicrobial susceptibility testing and esbl detection
antimicrobial susceptibilities were determined by the disk diffusion method on mueller-hinton agar  according to the guidelines of the comité de l’antibiogramme de la société française de microbiologie.
the following antibiotics were tested: amoxicillin, amoxicillin-clavulanate, ticarcillin, cephalotin, cefamandole, cefoxitin, cefotaxime, ceftazidime, imipenem, gentamicin, tobramycin, netilmicin, amikacin, nalidixic acid, pefloxacin, ciprofloxacin and trimethoprim-sulfamethoxazole.
suspected esbls were confirmed by the double-disk synergy test.
e. coli atcc  <dig> and k. pneumoniae atcc  <dig> were used as quality control strains.
fingerprinting analysis
after dna extraction by using the qiagen mini kit , repetitive extragenic palindromic  and enterobacterial repetitive intergenic consensus sequence pcr  were performed with the rep-1r, rep-2 t and eric- <dig> primers, respectively, as previously described
<cit> .
pattern profiles were considered different when at least one band differed.
molecular characterization of resistance genes
dna was extracted by the boiling method.
esbl-encoding genes were identified using specific primers for the blatem, blashv, blactx-m and blaoxa genes, previously described
<cit> , and followed by dna sequencing.
other blactx-m-15-associated antibiotic resistance genes -ib,qnra, qnrb, qnrs, teta, sul <dig> and sul2) were screened by pcr
<cit> .
all positive isolates for the aac-ib gene were further analyzed by digesting the purified pcr products with btsci  to identify aac-ib-cr, which lacks the btsci restriction site present in the wild-type gene
<cit> .
the upstream sequence of the blactx-m genes was explored by pcr and sequenced to detect isecp <dig>  the integrase gene  was detected by pcr using specific primers
<cit> .
the variable region of each class  <dig> integron was amplified using specific primers for the 5′ conserved segment  and 3′ conserved segment
<cit> , and gene cassettes were sequenced.
blastn was used to compare the sequences obtained to those present in the genbank database .
resistance transfer assays
conjugations were carried out in trypticase soy broth , with e. coli j53- <dig>  as the recipient.
mating broths were incubated at 37°c for 18 hr.
transconjugants were selected on drigalski agar plates  containing rifampicin  and cefotaxime .
transfer experiments using electroporation were performed for non-conjugative plasmids.
plasmid dna from donors was extracted with a qiagen plasmid midi kit .
purified plasmids were used to transform e. coli dh10b  by electroporation following the manufacturer’s instructions .
transformants were incubated at 37°c for  <dig>  hr and then selected on drigalski agar  supplemented with  <dig>  μg/ml cefotaxime.
transconjugants and transformants were tested for esbl production followed by pcr amplification of the esbl genes and plasmid replicon typing.
plasmid replicon type determination
plasmid replicons from transconjugants and transformants were determined using the pcr-based replicon typing method described previously by carattoli et al.
eighteen pairs of primers targeting the fia, fib, fic, hi <dig>  hi <dig>  i <dig>  l/m, n, p, w, t, a/c, k, b/o, x, y, f and fii replicons were used in single or multiplex pcr
<cit> .
phylogenetic group and virulence genotyping of e. coli
the phylogenetic groups of the e. coli isolates were determined by pcr,
<cit> , using a combination of three dna gene markers .
all isolates belonging to group b <dig> were analyzed by duplex pcr targeting the pabb and trpa genes to determine whether the isolate was a member of the o25b-st <dig> clonal group or not
<cit> .
the presence of  <dig> virulence factors found in expec was investigated by pcr with primers reported previously
<cit> .
these factors included fimh , sfa/foc , papg alleles , afa , hlya , cnf , fyua , iuta , kpsmii , trat , sat , iron  and iha .
RESULTS
description of the bacterial isolates
during the study period, we collected  <dig> isolates, of which  <dig> from hospitalized patients and  <dig> from patients attending the pasteur institute medical laboratory.
among these,  <dig> were identified as e. coli , k. pneumoniae , k. oxytoca  or e. cloacae  and  <dig> were esbl-producers of which  <dig> were selected for in-depth analysis.
inclusion criteria were: i) one isolate per patient; ii) only the referent isolate, in cases of a hospital outbreak; and iii) at least one isolate from every ward participating in the study.
among the  <dig> esbl-producing isolates,  <dig> were isolated from patients referred to the pasteur institute medical laboratory and  <dig> were from hospitalized patients.
distribution of isolates by hospital, ward and specimen is shown in table
<dig>
*other: csf, sputum.
ipm: pasteur institute medical laboratory.
hjra: joseph ravoahangy andrianavalona hospital.
homi: military hospital.
antimicrobial susceptibility analyses showed that all isolates were resistant to all the β-lactams used but were susceptible to cefoxitin and imipenem.
resistance to cefoxitin in all e. cloacae isolates was due to the inducible production of ampc β-lactamase from a chromosomal gene.
all esbl-producing isolates were also multidrug-resistant and most of them were resistant to: aminoglycosides , trimethoprim-sulfamethoxazole  and quinolones .
molecular epidemiology
eric-pcr and rep-pcr analyses revealed different restriction patterns for each isolate and showed that they were not clonally related .
molecular analysis
nucleotide sequence analysis of the blactx-m and blashv genes showed that only the ctx-m- <dig> and shv- <dig> genes were present in these isolates.
only tem- <dig> and oxa- <dig> were identified in the tem- and oxa-producing isolates.
the ctx-m- <dig> gene was detected in  <dig> isolates  and the shv- <dig> gene in  <dig> .
the isecp <dig> insertion sequence was identified in all  <dig> blactx-m-carrying isolates.
of the  <dig> isolates positive for ctx-m- <dig>  ten  also carried only tem- <dig>  nine  also carried only oxa- <dig>  and  <dig>  carried tem- <dig> and oxa- <dig> genes .
of the  <dig> shv-12-positive isolates, six  also carried only tem- <dig>  four  also carried only oxa- <dig> and six  carried tem- <dig> and oxa- <dig> genes .
eight isolates   carried both blactxm- <dig> and blashv- <dig> and six of these were additionally tem-1- and oxa-1-positive.
the resistance genes most frequently present were aac-ib  -ib-cr,  <dig> %), sul <dig> and sul <dig> , teta , qnrb  and qnra .
among the six isolates carrying blactxm- <dig>  blashv- <dig>  blatem- <dig> and blaoxa- <dig>  all of these also carried aac-ib -ib-cr), sul1-sul <dig>  and five harbored teta.
overall β-lactam resistant isolates harbored β-lactamases genes  as well as trimethoprim-sulfamethoxazole resistant isolates sulfamide genes .
ten  of ciprofloxacin resistant isolates and  <dig>  of ciprofloxacin susceptible isolates were qnr positive.
twenty five  of ciprofloxacin resistant isolates and  <dig>  of ciprofloxacin susceptible isolates were aac-ib-cr positive and,  <dig>  of amikacin susceptible isolates and  <dig>  of amikacin resistant isolates were aac-ib positive.
forty-eight isolates were positive for the class- <dig> integron gene and it was absent in only one k. oxytoca isolate.
we amplified the class  <dig> integrons in twenty-five  of these  <dig> isolates using 5′cs and 3′cs primers.
the sizes of the class  <dig> integron amplicons, which correspond to the approximate sizes of the cassette regions, were between  <dig>  kb and 2 kb.
seven different cassettes were identified, including the dfr gene that encodes resistance to trimethoprim and the aada gene that encodes resistance to streptomycin.
the two genes most frequently associated with each other were dfra <dig> and aada <dig>  .
resistance transfer
transfer of esbl by conjugation to e. coli j53- <dig> was successful for  <dig>  of the  <dig> esbl isolates, which consisted of eight e. coli, eight e. cloacae and 12 k. pneumoniae isolates and one k. oxytoca isolate.
esbl transfer by plasmid dna electroporation into e. coli dh10b was successful for five  of the  <dig> remaining isolates; four were e. coli isolates and one was a k. pneumoniae isolate.
the presence of blactx-m, blashv, blatem and blaoxa was confirmed by pcr in the  <dig> transconjugants and transformants.
transfers of non-esbl resistance genes  were also detected by antimicrobial susceptibility testing.
plasmid replicon type determination
pcr-based replicon typing in the  <dig> transconjugants and transformants demonstrated the presence of the incfii, hi <dig> and fia replicons in these isolates .
incfii was the most prevalent replicon type and was detected in  <dig>   of the  <dig> isolates.
hi <dig> was found in  <dig>  isolates  and fia was found in one e. coli isolate.
the plasmids carrying blactx-m- <dig> were assigned to the fii  and hi <dig>  replicon types.
plasmids carrying blashv- <dig>  or carrying both blactx-m- <dig> and blashv- <dig>  were assigned to fii.
*deep pus, surgical wounds.
e. coli phylogenetic groups and virulence factors
phylogenetic analysis of the  <dig> e. coli isolates revealed four main phylogenetic groups .
most of these isolates belonged to group a <dig> ,  <dig> of which were isolated from pus.
all  <dig> isolates harbored genes related to complement resistance  but none harbored any of the papg alleles or the fimh, afa, hlya, cnf <dig>  kpsmii or sat genes.
ten isolates from groups a <dig>  b <dig> and d harbored genes encoding siderophores  .
the single e. coli isolate in the b <dig> group was an o25b-st <dig> clone and was isolated from the urine of a hospitalized patient.
this e. coli isolate harbored blactx-m- <dig>  teta, aac-ib-cr and sul1-sul <dig>  and was assigned to the fii replicon type.
genes encoding siderophore  and genes involved in the formation of adhesins  or fimbriae  were detected in this isolate, but it produced neither cytotoxin nor hemolysin.
discussion
we extensively characterized  <dig> esbl-producing enterobacteriaceae collected over a period of 15 months in four hospitals and at the pasteur institute medical laboratory.
previous studies in antananarivo have shown resistant bacteria clonal diffusion in hospital settings
<cit> , but among the  <dig> non-representative esbl-producing enterobacteriaceae studied, no clonal isolates have been found.
the blactx-m- <dig> esbl gene is considered to be the most prevalent esbl worldwide
<cit> .
we also found blactx-m- <dig> to be the most prevalent esbl in madagascar, as it was detected in  <dig> % of the isolates we studied.
a study involving nine asian countries reported that blactx-m- <dig> was highly prevalent among esbl-producing k. pneumoniae isolates
<cit> .
in tunisia, dahmen et al.
reported that 91% of the esbl-producing isolates carried blactx-m- <dig> genes
<cit> .
our findings are intermediate between those found in asia and in tunisia and confirm the predominance of blactx-m- <dig> among esbl-producing isolates.
in antananarivo, a previous study conducted in the neonatal units of two hospitals in  <dig> documented that a clonal outbreak of k. pneumoniae harbored blactx-m- <dig> and blashv- <dig> genes
<cit> .
in  <dig>  a community-based study of the intestinal carriage of  <dig> esbl-producing enterobacteriaceae demonstrated that the most prevalent esbl gene was blactx-m- <dig> , followed by blactx-m- <dig>  blashv- <dig> and blashv-2a <cit> .
the presences of blactx-m- <dig>  blactx-m- <dig>  blashv- <dig> and blashv- <dig> is not surprising as molecular analysis indicated that blactx-m- <dig> derived from blactx-m- <dig> <cit>  and blashv- <dig> from blashv- <dig> <cit> .
ctx-m genes may disseminate through clonal expansion or horizontal gene transfer
<cit> .
in our study, isecp <dig> was found upstream from blactx-m- <dig> at variable distances, as was previously described
<cit> .
isecp <dig> was found to be in the vicinity of many blactx-m genes  and was reported to contain sequences resembling a typical promoter region
<cit> .
then, plasmids carrying blactx-m- <dig> were assigned to the incfii, incfia or inchi <dig> incompatibility group replicons.
association of the blactx-m- <dig> gene with incf plasmids carrying the fii replicon in association with the fia or fib replicon has been reported previously for isolates in canada, france, spain, tunisia, and the united kingdom
<cit> .
the first evidence of the association of the fii plasmid with the blactx-m- <dig> gene was demonstrated by sequencing the entire pc15-1a plasmid from epidemic e. coli isolated in canada
<cit> .
the inchi <dig> plasmid, frequently associated with blactx-m- <dig> or blactx-m- <dig>  was first identified in serratia marcescens <cit> , but rarely reported in association with blactx-m- <dig>
like blactx-m- <dig>  blashv- <dig> is also widely distributed.
in our study, 38% of the isolates harbored blashv- <dig>  first described in switzerland
<cit>  and subsequently found in various continents, including africa
<cit> , blashv- <dig> is most often found in asia
<cit> .
plasmids carrying blashv- <dig> were assigned to the incfii replicon, as previously reported in france
<cit> .
evolutionary analysis of genbank sequences indicated that blashv- <dig> evolved from the branch of blashv-2a <cit> .
although it is possible that this transformation occurred in antananarivo, as blashv-2a was reported in neonatal units in 2009
<cit> .
it can also be assumed that the local emergence of blashv- <dig> could be explained by introduction of international clones.
our antimicrobial susceptibility analysis of the esbl-producing isolates found highly prevalent resistances to gentamicin ; tobramycin ; ciprofloxacin  and to trimethoprim-sulfamethoxazole  and confirm the presence of multidrug-resistant isolates in antananarivo
<cit> .
the finding of multidrug resistance among esbl-producing isolates is of great clinical relevance due to the severely limited therapeutic options and the high risk of treatment failure in patients infected with these strains.
genes encoding esbls are often associated with determinants of resistance to other antimicrobial agents, including aminoglycosides -ib), fluoroquinolones , tetracycline , and trimethoprim-sulfamethoxazole  and are frequently located on plasmids belonging to the incf group
<cit> .
in this study, we found the first example in madagascar of the plasmid-mediated quinolone resistance  genes: qnrb  and qnra , and a variant gene aac-ib-cr  that encodes an aminoglycoside-modifying enzyme.
qnr gene prevalence was higher in the k. pneumoniae  isolates than in the e. coli  isolates, which has been noted by other authors
<cit> .
the aac-ib-cr gene accounted for  <dig> %  of the aac-ib genes detected.
this high proportion of aac-ib-cr/aac-ib was also observed in a previous study
<cit> .
the pmqr genes qnr and aac-ib-cr are now recognized to be geographically widespread
<cit> .
these genes have been previously reported to be associated with esbls.
the horizontal transfer of plasmids harboring genes encoding for esbls and pmqr genes could have promoted this co-resistance.
the cassette region could not be amplified by pcr in  <dig> class  <dig> integron-containing isolates, which may have been due to the lack of the 3′cs.
the analysis of  <dig> cassette regions revealed a predominance of aada and dfra genes, which confer resistance to aminoglycosides and trimethoprim, respectively.
this result correlates with previous studies of african enterobacteriaceae isolates
<cit> .
the combination of dfra17-aada <dig>  was the one most frequently detected in our study.
similar findings were reported for isolates from taiwan and tunisia, as dfra17-aada <dig> was found in  <dig> of  <dig>  and in  <dig> of  <dig>  e. coli class  <dig> integrons, respectively
<cit> .
analysis of the phylogenetic groups and virulence factors of e. coli isolates revealed that most of these isolates belong to group a <dig>  the phylogenetic group a <dig> consists of commensal enteric e. coli and may therefore be the natural reservoir of pathogenic isolates.
pathogenic e. coli isolates may have derived from commensal isolates by acquiring chromosomal or extra chromosomal virulence operons
<cit> .
although virulence determinants are considered to be mobile, strain phylogeny and virulence may be linked
<cit> .
the b <dig> phylogenetic group, which diverges from the commensal isolates, evolved toward extra intestinal virulence by acquiring numerous pathogenic determinants
<cit> .
we also encountered an e. coli isolate belonging to group b <dig>  harboring blactx-m- <dig> and other resistance genes, and corresponding to the worldwide pandemic clone o25b-st <dig>  it has been reported that most o25-st <dig> isolates are multidrug-resistant, produce ctx-m- <dig> esbl enzymes
<cit>  and harbor virulence genes required for pathogenic invasion of hosts.
in one study, the genes for adhesins , siderophores  and the toxin  were found in 95% - 100% of the o25b-st <dig> e. coli isolates
<cit> , but typical fimbriae and pilus genes, such as those encoded by the papa allele, were not.
in africa, few data exist on the presence of st <dig>  in a south african study, 43% of  <dig> isolates were st131
<cit> ; as were 50% of the ctx-m-15-producing e. coli isolates collected in the central african republic
<cit> .
the presence of this clone in antananarivo hospitals is of concern and further studies should be conducted to assess its prevalence.
CONCLUSIONS
our results highlight the dissemination of multidrug resistant enterobacteriaceae isolates in antananarivo, in different hospital settings and probably in the community.
these findings underline the need for a rational use of antibiotic and for appropriate methods of screening esbl in routine laboratories in antananarivo.
competing interest
the authors declare that they have no competing interests.
authors’ contributions
conception and design of the study and acquisition of data: hcr, fr, vr, at, ga. molecular and genetic studies, molecular analysis: hcr, ga. analysis of results: hcr, fr, vr, at, ga. draft of the manuscript: hcr, fr, bg, at, ga. revisiting of the manuscript for important intellectual content: vr, bg, at and ga. all authors have read and approved the final manuscript.

 extended-spectrum β-lactamase -producing bacteria represent a major worldwide threat among drug-resistant bacteria in both hospital and community settings. esbls are among the ambler classes a, confer resistance to β-lactam antibiotics except cephamycins and carbapenems, and are inhibited by clavulanic acid. esbls are often located on large plasmids that also harbor resistant genes to other antimicrobial classes with resulting multidrug-resistant isolates. the first esbls have evolved by genetic mutation from native β-lactamases tem and shv. recently, a novel type of esbls, the ctx-m enzymes, emerged worldwide, mostly from enterobacteriaceae. ctx-m β-lactamases are not closely related to tem or shv esbls but share high amino-acid identity with chromosomal β-lactamases from kluyvera spp.. now, blactx-m- is recognized as the most widely distributed ctx-m enzyme. it is derived from ctx-m- by a substitution of asp-240-gly which increases its catalytic efficiency against ceftazidime. blactx-m- are encoded on plasmids belonging to the incompatibility group incf. in the upstream region of ctx-m genes an insertion sequence element, isecp is commonly present and is likely responsible for the transposition process of the genes. e. coli is among the most prevalent causes of hospital-acquired and community-acquired bacterial infections and their resistances to antimicrobial agents have become a serious concern for healthcare providers. phylogenetic analyses have classified e. coli into four main phylogenetic groups. commensal isolates belong mainly to a and b groups whereas virulent extra-intestinal pathogenic e. coli are essentially from the b and d groups. expec harbor numerous virulence factors including α-hemolysin, cytotoxic necrotizing factor, adhesins and iron acquisition systems. the spread of blactx-m- has been mainly associated with the dissemination of a particular clone of e. coli st belonging to phylogenetic group b2. recently, an e. coli clone o st producing ctx-m- with high virulence potential and belonging to the b group, has been reported and represent a major public health problem. many reports have documented the emergence of esbl-producing enterobacteriaceae. in antananarivo, esbls were first detected in from uti in % of isolated enterobacteriaceae. in outbreaks of ctx-m- and shv-2-producing k. pneumoniae isolates have been described in two pediatric units. more recently, % of clinical isolates from patients in surgery and intensive care units and % of intestinal carriage isolates from children hospitalized in a pediatric department of a large teaching hospital were esbl-producers. for multidrug-resistant enterobacteriaceae isolates from antananarivo, we characterized: i) the genes encoding the esbls; ii) the drug resistance genes associated with the esbl genes; iii) gene cassettes present in the isolates; and iv) the plasmid incompatibility groups of the isolates. we also determined the phylogenetic groups and virulence factors of the e. coli isolates. methods ethical clearance the study protocols were approved by the national ethics committee of madagascar. written informed consents were obtained from all patients and at least one parent of each child before enrollment. patients between september and december a total of non-duplicate bacterial isolates were obtained from patients. patients were recruited from several wards in four hospitals in antananarivo, madagascar and patients referred to the pasteur institute medical laboratory in antananarivo. laboratory methods various clinical specimens were collected and submitted for bacterial analysis at the pasteur institute medical laboratory in antananarivo. presumptive enterobacteria isolates were identified using standard microbiological methods and the api 20e system. antimicrobial susceptibility testing and esbl detection antimicrobial susceptibilities were determined by the disk diffusion method on mueller-hinton agar according to the guidelines of the comité de l’antibiogramme de la société française de microbiologie. the following antibiotics were tested: amoxicillin, amoxicillin-clavulanate, ticarcillin, cephalotin, cefamandole, cefoxitin, cefotaxime, ceftazidime, imipenem, gentamicin, tobramycin, netilmicin, amikacin, nalidixic acid, pefloxacin, ciprofloxacin and trimethoprim-sulfamethoxazole. suspected esbls were confirmed by the double-disk synergy test. e. coli atcc and k. pneumoniae atcc were used as quality control strains. fingerprinting analysis after dna extraction by using the qiagen mini kit, repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence pcr were performed with the rep-1r, rep-2 t and eric- primers, respectively, as previously described. pattern profiles were considered different when at least one band differed. molecular characterization of resistance genes dna was extracted by the boiling method. esbl-encoding genes were identified using specific primers for the blatem, blashv, blactx-m and blaoxa genes, previously described, and followed by dna sequencing. other blactx-m-15-associated antibiotic resistance genes -ib,qnra, qnrb, qnrs, teta, sul and sul2) were screened by pcr. all positive isolates for the aac-ib gene were further analyzed by digesting the purified pcr products with btsci to identify aac-ib-cr, which lacks the btsci restriction site present in the wild-type gene. the upstream sequence of the blactx-m genes was explored by pcr and sequenced to detect isecp the integrase gene was detected by pcr using specific primers. the variable region of each class integron was amplified using specific primers for the 5′ conserved segment and 3′ conserved segment, and gene cassettes were sequenced. blastn was used to compare the sequences obtained to those present in the genbank database. resistance transfer assays conjugations were carried out in trypticase soy broth, with e. coli j53- as the recipient. mating broths were incubated at 37°c for 18 hr. transconjugants were selected on drigalski agar plates containing rifampicin and cefotaxime. transfer experiments using electroporation were performed for non-conjugative plasmids. plasmid dna from donors was extracted with a qiagen plasmid midi kit. purified plasmids were used to transform e. coli dh10b by electroporation following the manufacturer’s instructions. transformants were incubated at 37°c for  hr and then selected on drigalski agar supplemented with  μg/ml cefotaxime. transconjugants and transformants were tested for esbl production followed by pcr amplification of the esbl genes and plasmid replicon typing. plasmid replicon type determination plasmid replicons from transconjugants and transformants were determined using the pcr-based replicon typing method described previously by carattoli et al. eighteen pairs of primers targeting the fia, fib, fic, hi hi i l/m, n, p, w, t, a/c, k, b/o, x, y, f and fii replicons were used in single or multiplex pcr. phylogenetic group and virulence genotyping of e. coli the phylogenetic groups of the e. coli isolates were determined by pcr,, using a combination of three dna gene markers. all isolates belonging to group b were analyzed by duplex pcr targeting the pabb and trpa genes to determine whether the isolate was a member of the o25b-st clonal group or not. the presence of virulence factors found in expec was investigated by pcr with primers reported previously. these factors included fimh, sfa/foc, papg alleles, afa, hlya, cnf, fyua, iuta, kpsmii, trat, sat, iron and iha.  description of the bacterial isolates during the study period, we collected isolates, of which from hospitalized patients and from patients attending the pasteur institute medical laboratory. among these, were identified as e. coli, k. pneumoniae, k. oxytoca or e. cloacae and were esbl-producers of which were selected for in-depth analysis. inclusion criteria were: i) one isolate per patient; ii) only the referent isolate, in cases of a hospital outbreak; and iii) at least one isolate from every ward participating in the study. among the esbl-producing isolates, were isolated from patients referred to the pasteur institute medical laboratory and were from hospitalized patients. distribution of isolates by hospital, ward and specimen is shown in table  *other: csf, sputum. ipm: pasteur institute medical laboratory. hjra: joseph ravoahangy andrianavalona hospital. homi: military hospital. antimicrobial susceptibility analyses showed that all isolates were resistant to all the β-lactams used but were susceptible to cefoxitin and imipenem. resistance to cefoxitin in all e. cloacae isolates was due to the inducible production of ampc β-lactamase from a chromosomal gene. all esbl-producing isolates were also multidrug-resistant and most of them were resistant to: aminoglycosides, trimethoprim-sulfamethoxazole and quinolones. molecular epidemiology eric-pcr and rep-pcr analyses revealed different restriction patterns for each isolate and showed that they were not clonally related. molecular analysis nucleotide sequence analysis of the blactx-m and blashv genes showed that only the ctx-m- and shv- genes were present in these isolates. only tem- and oxa- were identified in the tem- and oxa-producing isolates. the ctx-m- gene was detected in isolates and the shv- gene in. the isecp insertion sequence was identified in all blactx-m-carrying isolates. of the isolates positive for ctx-m- ten also carried only tem- nine also carried only oxa- and carried tem- and oxa- genes. of the shv-12-positive isolates, six also carried only tem- four also carried only oxa- and six carried tem- and oxa- genes. eight isolates carried both blactxm- and blashv- and six of these were additionally tem-1- and oxa-1-positive. the resistance genes most frequently present were aac-ib -ib-cr, %), sul and sul, teta, qnrb and qnra. among the six isolates carrying blactxm- blashv- blatem- and blaoxa- all of these also carried aac-ib -ib-cr), sul1-sul and five harbored teta. overall β-lactam resistant isolates harbored β-lactamases genes as well as trimethoprim-sulfamethoxazole resistant isolates sulfamide genes. ten of ciprofloxacin resistant isolates and of ciprofloxacin susceptible isolates were qnr positive. twenty five of ciprofloxacin resistant isolates and of ciprofloxacin susceptible isolates were aac-ib-cr positive and, of amikacin susceptible isolates and of amikacin resistant isolates were aac-ib positive. forty-eight isolates were positive for the class- integron gene and it was absent in only one k. oxytoca isolate. we amplified the class integrons in twenty-five of these isolates using 5′cs and 3′cs primers. the sizes of the class integron amplicons, which correspond to the approximate sizes of the cassette regions, were between  kb and 2 kb. seven different cassettes were identified, including the dfr gene that encodes resistance to trimethoprim and the aada gene that encodes resistance to streptomycin. the two genes most frequently associated with each other were dfra and aada. resistance transfer transfer of esbl by conjugation to e. coli j53- was successful for of the esbl isolates, which consisted of eight e. coli, eight e. cloacae and 12 k. pneumoniae isolates and one k. oxytoca isolate. esbl transfer by plasmid dna electroporation into e. coli dh10b was successful for five of the remaining isolates; four were e. coli isolates and one was a k. pneumoniae isolate. the presence of blactx-m, blashv, blatem and blaoxa was confirmed by pcr in the transconjugants and transformants. transfers of non-esbl resistance genes were also detected by antimicrobial susceptibility testing. plasmid replicon type determination pcr-based replicon typing in the transconjugants and transformants demonstrated the presence of the incfii, hi and fia replicons in these isolates. incfii was the most prevalent replicon type and was detected in of the isolates. hi was found in isolates and fia was found in one e. coli isolate. the plasmids carrying blactx-m- were assigned to the fii and hi replicon types. plasmids carrying blashv- or carrying both blactx-m- and blashv- were assigned to fii. *deep pus, surgical wounds. e. coli phylogenetic groups and virulence factors phylogenetic analysis of the e. coli isolates revealed four main phylogenetic groups. most of these isolates belonged to group a, of which were isolated from pus. all isolates harbored genes related to complement resistance but none harbored any of the papg alleles or the fimh, afa, hlya, cnf kpsmii or sat genes. ten isolates from groups a b and d harbored genes encoding siderophores. the single e. coli isolate in the b group was an o25b-st clone and was isolated from the urine of a hospitalized patient. this e. coli isolate harbored blactx-m- teta, aac-ib-cr and sul1-sul and was assigned to the fii replicon type. genes encoding siderophore and genes involved in the formation of adhesins or fimbriae were detected in this isolate, but it produced neither cytotoxin nor hemolysin. discussion we extensively characterized esbl-producing enterobacteriaceae collected over a period of 15 months in four hospitals and at the pasteur institute medical laboratory. previous studies in antananarivo have shown resistant bacteria clonal diffusion in hospital settings, but among the non-representative esbl-producing enterobacteriaceae studied, no clonal isolates have been found. the blactx-m- esbl gene is considered to be the most prevalent esbl worldwide. we also found blactx-m- to be the most prevalent esbl in madagascar, as it was detected in % of the isolates we studied. a study involving nine asian countries reported that blactx-m- was highly prevalent among esbl-producing k. pneumoniae isolates. in tunisia, dahmen et al. reported that 91% of the esbl-producing isolates carried blactx-m- genes. our findings are intermediate between those found in asia and in tunisia and confirm the predominance of blactx-m- among esbl-producing isolates. in antananarivo, a previous study conducted in the neonatal units of two hospitals in documented that a clonal outbreak of k. pneumoniae harbored blactx-m- and blashv- genes. in a community-based study of the intestinal carriage of esbl-producing enterobacteriaceae demonstrated that the most prevalent esbl gene was blactx-m-, followed by blactx-m- blashv- and blashv-2a. the presences of blactx-m- blactx-m- blashv- and blashv- is not surprising as molecular analysis indicated that blactx-m- derived from blactx-m- and blashv- from blashv-. ctx-m genes may disseminate through clonal expansion or horizontal gene transfer. in our study, isecp was found upstream from blactx-m- at variable distances, as was previously described. isecp was found to be in the vicinity of many blactx-m genes and was reported to contain sequences resembling a typical promoter region. then, plasmids carrying blactx-m- were assigned to the incfii, incfia or inchi incompatibility group replicons. association of the blactx-m- gene with incf plasmids carrying the fii replicon in association with the fia or fib replicon has been reported previously for isolates in canada, france, spain, tunisia, and the united kingdom. the first evidence of the association of the fii plasmid with the blactx-m- gene was demonstrated by sequencing the entire pc15-1a plasmid from epidemic e. coli isolated in canada. the inchi plasmid, frequently associated with blactx-m- or blactx-m- was first identified in serratia marcescens, but rarely reported in association with blactx-m- like blactx-m- blashv- is also widely distributed. in our study, 38% of the isolates harbored blashv- first described in switzerland and subsequently found in various continents, including africa, blashv- is most often found in asia. plasmids carrying blashv- were assigned to the incfii replicon, as previously reported in france. evolutionary analysis of genbank sequences indicated that blashv- evolved from the branch of blashv-2a. although it is possible that this transformation occurred in antananarivo, as blashv-2a was reported in neonatal units in 2009. it can also be assumed that the local emergence of blashv- could be explained by introduction of international clones. our antimicrobial susceptibility analysis of the esbl-producing isolates found highly prevalent resistances to gentamicin; tobramycin; ciprofloxacin and to trimethoprim-sulfamethoxazole and confirm the presence of multidrug-resistant isolates in antananarivo. the finding of multidrug resistance among esbl-producing isolates is of great clinical relevance due to the severely limited therapeutic options and the high risk of treatment failure in patients infected with these strains. genes encoding esbls are often associated with determinants of resistance to other antimicrobial agents, including aminoglycosides -ib), fluoroquinolones, tetracycline, and trimethoprim-sulfamethoxazole and are frequently located on plasmids belonging to the incf group. in this study, we found the first example in madagascar of the plasmid-mediated quinolone resistance genes: qnrb and qnra, and a variant gene aac-ib-cr that encodes an aminoglycoside-modifying enzyme. qnr gene prevalence was higher in the k. pneumoniae isolates than in the e. coli isolates, which has been noted by other authors. the aac-ib-cr gene accounted for % of the aac-ib genes detected. this high proportion of aac-ib-cr/aac-ib was also observed in a previous study. the pmqr genes qnr and aac-ib-cr are now recognized to be geographically widespread. these genes have been previously reported to be associated with esbls. the horizontal transfer of plasmids harboring genes encoding for esbls and pmqr genes could have promoted this co-resistance. the cassette region could not be amplified by pcr in class integron-containing isolates, which may have been due to the lack of the 3′cs. the analysis of cassette regions revealed a predominance of aada and dfra genes, which confer resistance to aminoglycosides and trimethoprim, respectively. this result correlates with previous studies of african enterobacteriaceae isolates. the combination of dfra17-aada was the one most frequently detected in our study. similar findings were reported for isolates from taiwan and tunisia, as dfra17-aada was found in of and in of e. coli class integrons, respectively. analysis of the phylogenetic groups and virulence factors of e. coli isolates revealed that most of these isolates belong to group a the phylogenetic group a consists of commensal enteric e. coli and may therefore be the natural reservoir of pathogenic isolates. pathogenic e. coli isolates may have derived from commensal isolates by acquiring chromosomal or extra chromosomal virulence operons. although virulence determinants are considered to be mobile, strain phylogeny and virulence may be linked. the b phylogenetic group, which diverges from the commensal isolates, evolved toward extra intestinal virulence by acquiring numerous pathogenic determinants. we also encountered an e. coli isolate belonging to group b harboring blactx-m- and other resistance genes, and corresponding to the worldwide pandemic clone o25b-st it has been reported that most o25-st isolates are multidrug-resistant, produce ctx-m- esbl enzymes and harbor virulence genes required for pathogenic invasion of hosts. in one study, the genes for adhesins, siderophores and the toxin were found in 95% - 100% of the o25b-st e. coli isolates, but typical fimbriae and pilus genes, such as those encoded by the papa allele, were not. in africa, few data exist on the presence of st in a south african study, 43% of isolates were st131; as were 50% of the ctx-m-15-producing e. coli isolates collected in the central african republic. the presence of this clone in antananarivo hospitals is of concern and further studies should be conducted to assess its prevalence.  our results highlight the dissemination of multidrug resistant enterobacteriaceae isolates in antananarivo, in different hospital settings and probably in the community. these findings underline the need for a rational use of antibiotic and for appropriate methods of screening esbl in routine laboratories in antananarivo. competing interest the authors declare that they have no competing interests. authors’ contributions conception and design of the study and acquisition of data: hcr, fr, vr, at, ga. molecular and genetic studies, molecular analysis: hcr, ga. analysis of results: hcr, fr, vr, at, ga. draft of the manuscript: hcr, fr, bg, at, ga. revisiting of the manuscript for important intellectual content: vr, bg, at and ga. all authors have read and approved the final manuscript. 
all blactx-m- positive isolates also contained the isecp insertion element. our results highlight the dissemination of multidrug resistant enterobacteriaceae isolates in antananarivo. forty-nine e. coli, k. pneumoniae, k. oxytoca and e. cloacae esbl-producing isolates were studied. these findings underline the need for a rational use of antibiotic and for appropriate methods of screening esbl in routine laboratories in antananarivo. in antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. we investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase -producing enterobacteriaceae isolated in community settings and in hospitals in antananarivo, madagascar. phylogenetic grouping and virulence genotyping of the e. coli isolates revealed that most of them belonged to group a one isolate assigned to group b harbored blactx-m- and five virulence genes and was related to the o25b-st clone.
 we investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase -producing enterobacteriaceae isolated in community settings and in hospitals in antananarivo, madagascar.  forty-nine e. coli, k. pneumoniae, k. oxytoca and e. cloacae esbl-producing isolates were studied. in antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. gene amplification analysis and sequencing revealed that % of the isolates harbored blactx-m- and % harbored blashv- the non-esbls resistance genes detected were blatem- blaoxa- aac-ib,aac-ib-cr, teta, sul- sul- qnra, qnrb and catb- we found dfra and aada gene cassettes in the class integron variable regions of the isolates, and the combination of dfra17-aada to be the most prevalent. all blactx-m- positive isolates also contained the isecp insertion element. conjugation and transformation experiments indicated that % of the antibiotic resistance genes resided on plasmids. through a pcr based replicon typing method, plasmids carrying the blashv- or blactx-m- genes were assigned to either the incfii replicon type or, rarely, to the hi replicon type. all isolates were subtyped by the rep-pcr and eric-pcr methods. phylogenetic grouping and virulence genotyping of the e. coli isolates revealed that most of them belonged to group a one isolate assigned to group b harbored blactx-m- and five virulence genes and was related to the o25b-st clone.  our results highlight the dissemination of multidrug resistant enterobacteriaceae isolates in antananarivo. these findings underline the need for a rational use of antibiotic and for appropriate methods of screening esbl in routine laboratories in antananarivo. 