Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar

  • Hanitra C Rakotonirina1, 4Email author,

    Affiliated with

    • Benoît Garin1,

      Affiliated with

      • Frédérique Randrianirina1,

        Affiliated with

        • Vincent Richard1, 2,

          Affiliated with

          • Antoine Talarmin1, 3 and

            Affiliated with

            • Guillaume Arlet4, 5

              Affiliated with

              BMC Microbiology201313:85

              DOI: 10.1186/1471-2180-13-85

              Received: 19 November 2012

              Accepted: 22 March 2013

              Published: 17 April 2013

              Abstract

              Background

              We investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar.

              Results

              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 75.5% (n=37) of the isolates harbored bla CTX-M-15 and 38.7% (n=19) harbored bla SHV-12. The non-ESBLs resistance genes detected were bla TEM-1, bla OXA-1, aac(6 )-Ib, aac(6 )-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All bla CTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the bla SHV-12 or bla CTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 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 A1. One isolate assigned to group B2 harbored bla CTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone.

              Conclusions

              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.

              Background

              Extended-spectrum β-lactamase (ESBL)-producing bacteria represent a major worldwide threat among drug-resistant bacteria in both hospital and community settings [1]. ESBLs are among the Ambler classes A, confer resistance to β-lactam antibiotics except cephamycins and carbapenems, and are inhibited by clavulanic acid [1]. ESBLs are often located on large plasmids that also harbor resistant genes to other antimicrobial classes with resulting multidrug-resistant isolates [2].

              The first ESBLs have evolved by genetic mutation from native β-lactamases TEM and SHV [3][4]. Recently, a novel type of ESBLs, the CTX-M enzymes, emerged worldwide, mostly from Enterobacteriaceae[5, 6]. 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. [7]. Now, bla CTX-M-15 is recognized as the most widely distributed CTX-M enzyme [8]. It is derived from CTX-M-3 by a substitution of Asp-240-Gly which increases its catalytic efficiency against ceftazidime [9]. bla CTX-M-15 are encoded on plasmids belonging to the incompatibility group IncF [10]. In the upstream region of CTX-M genes an insertion sequence element, ISEcp1, is commonly present and is likely responsible for the transposition process of the genes [11].

              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 [5]. Phylogenetic analyses have classified E. coli into four main phylogenetic groups (A, B1, B2, and D). Commensal isolates belong mainly to A and B1 groups whereas virulent extra-intestinal pathogenic E. coli (ExPEC) are essentially from the B2 and D groups [12, 13]. ExPEC harbor numerous virulence factors including α-hemolysin, cytotoxic necrotizing factor, adhesins and iron acquisition systems [12]. The spread of bla CTX-M-15 has been mainly associated with the dissemination of a particular clone of E. coli ST131 belonging to phylogenetic group B2 [14, 15]. Recently, an E. coli clone O25 ST131, producing CTX-M-15, with high virulence potential and belonging to the B2 group, has been reported and represent a major public health problem [14, 15].

              Many reports have documented the emergence of ESBL-producing Enterobacteriaceae[1618]. In Antananarivo, ESBLs were first detected in 2005 from UTI in 9.7% of isolated Enterobacteriaceae[19]. In 2006, outbreaks of CTX-M-15 and SHV-2-producing K. pneumoniae isolates have been described in two pediatric units [20]. More recently, 21.3% of clinical isolates from patients in surgery and intensive care units [21] and 21.2% of intestinal carriage isolates from children hospitalized in a pediatric department of a large teaching hospital [22] were ESBL-producers.

              For 49 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 2006 and December 2007, a total of 909 non-duplicate bacterial isolates were obtained from 909 patients. 830 patients were recruited from several wards in four hospitals in Antananarivo, Madagascar (two national university teaching hospitals: Joseph Ravoahangy Andrianavalona Hospital and Befelatanana Hospital; a military hospital: Soavinandriana Hospital; and a pediatric hospital: Tsaralalana Hospital) and 79 patients referred to the Pasteur Institute Medical Laboratory in Antananarivo.

              Laboratory methods

              Various clinical specimens (including blood-culture, urine, pus, sputum and CSF) 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 (Bio-Mérieux SA, Marcy l’Etoile, France).

              Antimicrobial susceptibility testing and ESBL detection

              Antimicrobial susceptibilities were determined by the disk diffusion method on Mueller-Hinton agar (Bio-Rad, Marne la Coquette, France) 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 25922 and K. pneumoniae ATCC 700603 were used as quality control strains.

              Fingerprinting analysis

              After DNA extraction by using the Qiagen Mini kit (Qiagen, Courtaboeuf, France), repetitive extragenic palindromic (Rep-PCR) and Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were performed with the rep-1R, rep-2 T and ERIC-2 primers, respectively, as previously described [18]. 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 bla TEM, bla SHV, bla CTX-M and bla OXA genes, previously described [23], and followed by DNA sequencing. Other bla CTX-M-15-associated antibiotic resistance genes (i.e., aac(6 )-Ib, qnrA, qnrB, qnrS, tetA, sul1 and sul2) were screened by PCR [24, 25]. All positive isolates for the aac(6 )-Ib gene were further analyzed by digesting the purified PCR products with BtsCI (New England Biolabs, Beverly, MA) to identify aac(6 )-Ib-cr, which lacks the BtsCI restriction site present in the wild-type gene [26]. The upstream sequence of the bla CTX-M genes was explored by PCR and sequenced to detect ISEcp1. The integrase gene (int1) was detected by PCR using specific primers [27]. The variable region of each class 1 integron was amplified using specific primers for the 5 conserved segment (5CS) and 3 conserved segment (3CS) [27], and gene cassettes were sequenced. BlastN was used to compare the sequences obtained to those present in the GenBank database (http://​blast.​ncbi.​nlm.​nih.​gov).

              Resistance transfer assays

              Conjugations were carried out in trypticase soy broth (Bio-Rad), with E. coli J53-2 (pro, met, Rifr) as the recipient. Mating broths were incubated at 37°C for 18 hr. Transconjugants were selected on Drigalski agar plates (Bio-Rad) containing rifampicin (250 μg/ml) and cefotaxime (2.5 μg/ml).

              Transfer experiments using electroporation were performed for non-conjugative plasmids. Plasmid DNA from donors was extracted with a QIAGEN plasmid midi kit (QIAGEN, Courtaboeuf, France). Purified plasmids were used to transform E. coli DH10B (Invitrogen SARL, Cergy-Pontoise, France) by electroporation following the manufacturer’s instructions (Bio-Rad). Transformants were incubated at 37°C for 1.5 hr and then selected on Drigalski agar (Bio-Rad) supplemented with 2.5 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, HI1, HI2, I1, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FII replicons were used in single or multiplex PCR [28].

              Phylogenetic group and virulence genotyping of E. coli

              The phylogenetic groups of the E. coli isolates were determined by PCR, [13], using a combination of three DNA gene markers (chuA, yjaA and TSPE4-C2). All isolates belonging to group B2 were analyzed by duplex PCR targeting the pabB and trpA genes to determine whether the isolate was a member of the O25b-ST131 clonal group or not [29].

              The presence of 15 virulence factors found in ExPEC was investigated by PCR with primers reported previously [16]. These factors included fimH (type 1 fimbriae), sfa/foc (S and F1C fimbriae), papG alleles (G adhesin classes of P fimbriae), afa (fimbrial adhesin), hlyA (alpha-haemolysin A), cnf (cytotoxic necrotizating factor 1), fyuA (genes of yersiniabactin), iutA (aerobactin receptor), kpsMII (group 2 capsules), traT (genes related to complement resistance), sat (secreted autotransporter toxin), IroN (iron related genes) and Iha (IrgA homologue adhesin).

              Results

              Description of the bacterial isolates

              During the study period, we collected 909 isolates, of which 830 from hospitalized patients and 79 from patients attending the Pasteur Institute medical laboratory. Among these, 262 were identified as E. coli (n=75), K. pneumoniae (n=95), K. oxytoca (n=12) or E. cloacae (n=80) and 239 were ESBL-producers of which 49 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 49 ESBL-producing isolates, 13 were isolated from patients referred to the Pasteur Institute Medical Laboratory and 36 were from hospitalized patients. Distribution of isolates by hospital, ward and specimen is shown in Table 1.
              Table 1

              Distribution of isolates among patient category, ward and specimen types

                  

              Hospital

              Ward

              Specimen

              Species

              No

              Hospital

              IPM

              HJRA

              HOMI

              Befelatanana

              Tsaralalana

              Surgery

              Trauma

              Intensive care

              Pediatrics

              Urology

              Dermato

              Pus

              Blood

              Urine

              Other*

              E. cloacae

              14

              12

              2

              8

              2

              1

              1

              2

              5

              1

              3

              1

              0

              9

              4

              1

              0

              E. coli

              18

              14

              4

              12

              2

              0

              0

              3

              6

              3

              0

              1

              1

              12

              0

              4

              2

              K. pneumoniae

              14

              7

              7

              4

              3

              0

              0

              1

              3

              3

              0

              0

              0

              6

              3

              5

              0

              K. oxytoca

              3

              3

              0

              0

              1

              1

              1

              0

              0

              1

              2

              0

              0

              0

              3

              0

              0

              No (%)

              49 (%)

              36 (73.5)

              13 (26.5)

              24 (49)

              8 (16.3)

              2 (4.1)

              2 (4.1)

              6 (12.2)

              14 (28.6)

              8 (16.3)

              5 (10.2)

              2 (4.1)

              1 (2)

              27 (55.1)

              10 (20.4)

              10 (20.4)

              2 (4.1)

              *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 (87.7% to gentamicin, 93.8% to tobramycin), trimethoprim-sulfamethoxazole (100%) and quinolones (75.5% to nalidixic acid, 69.3% to ciprofloxacin).

              Molecular epidemiology

              ERIC-PCR and rep-PCR analyses revealed different restriction patterns for each isolate and showed that they were not clonally related (data not shown).

              Molecular analysis

              Nucleotide sequence analysis of the bla CTX-M and bla SHV genes showed that only the CTX-M-15 and SHV-12 genes were present in these isolates. Only TEM-1 and OXA-1 were identified in the TEM- and OXA-producing isolates.

              The CTX-M-15 gene was detected in 37 isolates (75.5%) and the SHV-12 gene in 19 (38%). The ISEcp1 insertion sequence was identified in all 37 bla CTX-M-carrying isolates. Of the 37 isolates positive for CTX-M-15, ten (27%) also carried only TEM-1, nine (24.3%) also carried only OXA-1, and 16 (43.2%) carried TEM-1 and OXA-1 genes (Table 1). Of the 19 SHV-12-positive isolates, six (31.6%) also carried only TEM-1, four (20.1%) also carried only OXA-1 and six (31.6%) carried TEM-1 and OXA-1 genes (Table 1). Eight isolates (16.3%) (two E. coli, five K. pneumoniae and one E. cloacae) carried both bla CTXM-15 and bla SHV-12 and six of these were additionally TEM-1- and OXA-1-positive.

              The resistance genes most frequently present were aac(6 )-Ib (n=35, 71.4%) (33 were aac(6 )-Ib-cr, 67.3%), sul1 and sul2 (n=25, 51%), tetA (n=24, 48.9%), qnrB (n=12, 24.5%) and qnrA (n=1, 2%). Among the six isolates carrying bla CTXM-15, bla SHV-12, bla TEM-1 and bla OXA-1, all of these also carried aac(6 )-Ib (5 were aac(6 )-Ib-cr), sul1-sul2, and five harbored tetA.

              Overall β-lactam resistant isolates harbored β-lactamases genes (CTX-M-15, SHV-12, TEM-1 and/or OXA-1) as well as trimethoprim-sulfamethoxazole resistant isolates sulfamide genes (sul1 and/or sul2). Ten (27.8%) of ciprofloxacin resistant isolates and 3 (25%) of ciprofloxacin susceptible isolates were qnr positive. Twenty five (69.2%) of ciprofloxacin resistant isolates and 8 (61.5%) of ciprofloxacin susceptible isolates were aac(6 )-Ib-cr positive And, 27 (71%) of amikacin susceptible isolates and 8 (72.7%) of amikacin resistant isolates were aac(6 )-Ib positive.

              Forty-eight isolates were positive for the class-1 integron gene and it was absent in only one K. oxytoca isolate. We amplified the class 1 integrons in twenty-five (52%) of these 48 isolates using 5CS and 3CS primers. The sizes of the class 1 integron amplicons, which correspond to the approximate sizes of the cassette regions, were between 0.7 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 dfrA17 and aadA5 (11/25, 22.4%) (Table 2).
              Table 2

              Characteristics of ESBL-producing Enterobacteriaceae isolates and their associated drug resistance genes and gene cassettes

                

              ESBLs

              Other β-lactamases

              Associated drug resistance genes

              Gene cassettes

              Species

              No

              CTX-M-15

              SHV-12

              Both

              TEM-1

              OXA-1

              TetA

              aac6'-1b

              aac6'-1b-cr

              qnrA

              qnrB

              catB3

              sul1

              sul2

              sul1- sul2

              aadA1

              aadA2

              aadA4

              aadA5

              dfrA5

              drA22

              dfrA17-aadA5

              E. coli

              18

              14

              2

              2

              12

              13

              8

              14

              13

              0

              3

              0

              2

              3

              8

              2

              1

              1

              1

              2

              0

              6

              K. pneumoniae

              14

              6

              3

              5

              7

              13

              9

              13

              13

              0

              5

              4

              2

              5

              7

              0

              2

              0

              0

              1

              1

              3

              K. oxytoca

              3

              1

              2

              0

              1

              0

              0

              0

              0

              0

              0

              0

              0

              0

              2

              0

              0

              0

              1

              0

              0

              0

              E. cloacae

              14

              8

              4

              1

              12

              2

              7

              8

              7

              1

              4

              0

              0

              6

              8

              0

              1

              1

              0

              0

              0

              2

              Totals

              49

              29

              11

              8

              32

              28

              24

              35

              33

              1

              12

              4

              4

              14

              25

              2

              4

              2

              2

              3

              1

              11

              Resistance transfer

              Transfer of ESBL by conjugation to E. coli J53-2 was successful for 29 (59.2%) of the 49 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 (10.2%) of the 20 remaining isolates; four were E. coli isolates and one was a K. pneumoniae isolate. The presence of bla CTX-M, bla SHV, bla TEM and bla OXA was confirmed by PCR in the 34 transconjugants and transformants. Transfers of non-ESBL resistance genes (tetracycline, gentamicin and trimethoprim-sulfamethoxazole) were also detected by antimicrobial susceptibility testing.

              Plasmid replicon type determination

              PCR-based replicon typing in the 34 transconjugants and transformants demonstrated the presence of the IncFII, HI2 and FIA replicons in these isolates (Table 3). IncFII was the most prevalent replicon type and was detected in 20 (58.8%) (10 E. coli and 10 K. pneumoniae) of the 34 isolates. HI2 was found in 13 (38.2%) isolates (eight E. cloacae, three K. pneumoniae, one E. coli and one K. oxytoca) and FIA was found in one E. coli isolate. The plasmids carrying bla CTX-M-15 were assigned to the FII (n=12) and HI2 (n=8) replicon types. Plasmids carrying bla SHV-12 (n=5) or carrying both bla CTX-M-15 and bla SHV-12 (n=2) were assigned to FII.
              Table 3

              β-lactamase genes transferred to transconjugants and electroporants and their replicon type

              β-lactamase genes

              Replicon type

              Transconjugants

               

              Electroporants

               

              E. coli

              K. pneumoniae

              K. oxytoca

              E. cloacae

              Totals

              E. coli

              K. pneumoniae

              Totals

              CTX-M-15

              FII

              2

              1

              0

              0

              3

              2

              0

              2

               

              HI2

              0

              1

              0

              1

              2

              0

              0

               
               

              FIA/FIB

              1

              0

              0

              0

              1

              0

              0

               

              SHV-12

              FII

              0

              3

              0

              0

              3

              1

              0

              1

              OXA-1

              FII

              0

              1

              0

              0

              1

              0

              0

               

              CTX-M-15+TEM-1

              FII

              0

              0

              0

              0

               

              1

              0

              1

               

              HI2

              0

              0

              1

              6

              7

              0

              0

               

              CTX-M-15+OXA-1

              FII

              3

              3

              0

              0

              6

              0

              0

               
               

              HI2

              1

              1

              0

              0

              2

              0

              0

               

              SHV-12+TEM-1

              FII

              1

              0

              0

              0

              1

              0

              0

               

              TEM-1+OXA-1

              HI2

              0

              0

              0

              1

              1

              0

              0

               

              CTX-M-15+SHV-12+TEM-1

              FII

              0

              0

              0

              0

               

              0

              1

              1

              CTX-M-15+TEM-1+OXA-1

              HI2

              0

              1

              0

              0

              1

              0

              0

               

              CTX-M-15+SHV-12+OXA-1

              FII

              0

              1

              0

              0

              1

              0

              0

               

              Totals

               

              8

              12

              1

              8

              29

              4

              1

              5

              Table 4

              Phylogenetic and virulence factors in the E. coli isolates

              Phylogenetic

              No

              Specimen

              Virulence factor

              group

               

              Pus*

              Urine

              Sputum

              CSF

              fyuA

              iutA

              sfa

              IroN

              Iha

              traT

              A1

              14

              11

              1

              1

              1

              3

              6

              0

              2

              0

              14

              B1

              2

              1

              1

              0

              0

              1

              1

              0

              0

              0

              2

              B2

              1

              0

              1

              0

              0

              1

              1

              1

              0

              1

              1

              D1

              1

              0

              1

              0

              0

              1

              1

              0

              0

              0

              1

              Totals

              18

              12

              4

              1

              1

              6

              9

              1

              2

              1

              18

              *Deep pus, surgical wounds.

              E. coli phylogenetic groups and virulence factors

              Phylogenetic analysis of the 18 E. coli isolates revealed four main phylogenetic groups (A1, B1, B2 and D). Most of these isolates belonged to group A1 (77.7%, n=14), 11 of which were isolated from pus. All 18 isolates harbored genes related to complement resistance (traT) but none harbored any of the papG alleles or the fimH, afa, hlyA, cnf1, kpsMII or sat genes. Ten isolates from groups A1, B1 and D harbored genes encoding siderophores (fyuA, iutA and IroN) (Table 4).

              The single E. coli isolate in the B2 group was an O25b-ST131 clone and was isolated from the urine of a hospitalized patient. This E. coli isolate harbored bla CTX-M-15, tetA, aac(6 )-Ib-cr and sul1-sul2, and was assigned to the FII replicon type. Genes encoding siderophore (fyuA and iutA) and genes involved in the formation of adhesins (iha) or fimbriae (sfa) were detected in this isolate, but it produced neither cytotoxin nor hemolysin.

              Discussion

              We extensively characterized 49 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 [20, 30], but among the 49 non-representative ESBL-producing Enterobacteriaceae studied, no clonal isolates have been found.

              The bla CTX-M-15 ESBL gene is considered to be the most prevalent ESBL worldwide [17, 18, 23, 31, 32]. We also found bla CTX-M-15 to be the most prevalent ESBL in Madagascar, as it was detected in 75.5% of the isolates we studied. A study involving nine Asian countries reported that bla CTX-M-15 was highly prevalent among ESBL-producing K. pneumoniae isolates (60%, 55/92) [17]. In Tunisia, Dahmen et al. reported that 91% of the ESBL-producing isolates carried bla CTX-M-15 genes [23]. Our findings are intermediate between those found in Asia and in Tunisia and confirm the predominance of bla CTX-M-15 among ESBL-producing isolates. In Antananarivo, a previous study conducted in the neonatal units of two hospitals in 2006 documented that a clonal outbreak of K. pneumoniae harbored bla CTX-M-15 and bla SHV-2 genes [20]. In 2009, a community-based study of the intestinal carriage of 49 ESBL-producing Enterobacteriaceae demonstrated that the most prevalent ESBL gene was bla CTX-M-15 (93.9%), followed by bla CTX-M-3, bla SHV-12 and bla SHV-2a[33]. The presences of bla CTX-M-15, bla CTX-M-3, bla SHV-2 and bla SHV-12 is not surprising as molecular analysis indicated that bla CTX-M-15 derived from bla CTX-M-3[6] and bla SHV-12 from bla SHV-2[34].

              CTX-M genes may disseminate through clonal expansion or horizontal gene transfer [35, 36]. In our study, ISEcp1 was found upstream from bla CTX-M-15 at variable distances, as was previously described [18]. ISEcp1 was found to be in the vicinity of many bla CTX-M genes (including bla CTX-M-15) and was reported to contain sequences resembling a typical promoter region [11]. Then, plasmids carrying bla CTX-M-15 were assigned to the IncFII, IncFIA or IncHI2 incompatibility group replicons. Association of the bla CTX-M-15 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 [35, 36]. The first evidence of the association of the FII plasmid with the bla CTX-M-15 gene was demonstrated by sequencing the entire pC15-1a plasmid from epidemic E. coli isolated in Canada [2]. The IncHI2 plasmid, frequently associated with bla CTX-M-2 or bla CTX-M-9, was first identified in Serratia marcescens[10], but rarely reported in association with bla CTX-M-15.

              Like bla CTX-M-15, bla SHV-12 is also widely distributed. In our study, 38% of the isolates harbored bla SHV-12. First described in Switzerland [37] and subsequently found in various continents, including Africa [38], bla SHV-12 is most often found in Asia [34]. Plasmids carrying bla SHV-12 were assigned to the IncFII replicon, as previously reported in France [39]. Evolutionary analysis of GenBank sequences indicated that bla SHV-12 evolved from the branch of bla SHV-2a[34]. Although it is possible that this transformation occurred in Antananarivo, as bla SHV-2a was reported in neonatal units in 2009 [20]. It can also be assumed that the local emergence of bla SHV-12 could be explained by introduction of international clones.

              Our antimicrobial susceptibility analysis of the ESBL-producing isolates found highly prevalent resistances to gentamicin (87.7%); tobramycin (93.8%); ciprofloxacin (69.3%) and to trimethoprim-sulfamethoxazole (100%) and confirm the presence of multidrug-resistant isolates in Antananarivo [19, 22]. 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 (aac(6)-Ib), fluoroquinolones (qnr), tetracycline (tetA), and trimethoprim-sulfamethoxazole (sul) and are frequently located on plasmids belonging to the IncF group [10]. In this study, we found the first example in Madagascar of the plasmid-mediated quinolone resistance (PMQR) genes: qnrB (24.5%) and qnrA (2%), and a variant gene aac(6 )-Ib-cr (67.3%) that encodes an aminoglycoside-modifying enzyme. Qnr gene prevalence was higher in the K. pneumoniae (41.7%) isolates than in the E. coli (25%) isolates, which has been noted by other authors [24, 40]. The aac(6 )-Ib-cr gene accounted for 94.3% (33/35) of the aac(6 )-Ib genes detected. This high proportion of aac(6 )-Ib-cr/aac(6 )-Ib was also observed in a previous study [40]. The PMQR genes qnr and aac(6 )-Ib-cr are now recognized to be geographically widespread [24, 25]. 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 23 class 1 integron-containing isolates, which may have been due to the lack of the 3CS. The analysis of 25 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 [27, 41]. The combination of dfrA17-aadA5 (22%) was the one most frequently detected in our study. Similar findings were reported for isolates from Taiwan and Tunisia, as dfrA17-aadA5 was found in 81 of 224 (36%) and in 3 of 4 (75%) E. coli class 1 integrons, respectively [42, 43].

              Analysis of the phylogenetic groups and virulence factors of E. coli isolates revealed that most of these isolates belong to group A1. The phylogenetic group A1 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 [44]. Although virulence determinants are considered to be mobile, strain phylogeny and virulence may be linked [45]. The B2 phylogenetic group, which diverges from the commensal isolates, evolved toward extra intestinal virulence by acquiring numerous pathogenic determinants [12].

              We also encountered an E. coli isolate belonging to group B2, harboring bla CTX-M-15 and other resistance genes, and corresponding to the worldwide pandemic clone O25b-ST131. It has been reported that most O25-ST131 isolates are multidrug-resistant, produce CTX-M-15 ESBL enzymes [14] and harbor virulence genes required for pathogenic invasion of hosts. In one study, the genes for adhesins (iha, fimH), siderophores (fyuA, iutA) and the toxin (sat) were found in 95% - 100% of the O25b-ST131 E. coli isolates [14], 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 ST131. In a South African study, 43% of 23 isolates were ST131 [46]; as were 50% of the CTX-M-15-producing E. coli isolates collected in the Central African Republic [13]. The presence of this clone in Antananarivo hospitals is of concern and further studies should be conducted to assess its prevalence.

              Conclusion

              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.

              Declarations

              Acknowledgements

              We thank Delphine Geneste and Nathalie Genel, for technical assistance, for participation in molecular studies. This study was performed with grants from Institut Pasteur de Madagascar and from Pierre and Marie Curie University.

              Authors’ Affiliations

              (1)
              Institut Pasteur de Madagascar
              (2)
              Institut Pasteur de Dakar
              (3)
              Institut Pasteur de Guadeloupe
              (4)
              Faculté de Médecine, Laboratoire de Bactériologie, Université Pierre et Marie Curie
              (5)
              Assistance Publique Hôpitaux de Paris, Hôpital Tenon, Laboratoire de Bactériologie

              References

              1. Bradford PA: Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001, 14:933–951. table of contentsPubMedView Article
              2. Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP: Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother 2004, 48:3758–3764.PubMedView Article
              3. Kliebe C, Nies BA, Meyer JF, Tolxdorff-Neutzling RM, Wiedemann B: Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrob Agents Chemother 1985, 28:302–307.PubMedView Article
              4. Sougakoff W, Goussard S, Gerbaud G, Courvalin P: Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase genes. Rev Infect Dis 1988, 10:879–884.PubMedView Article
              5. Pitout JD, Laupland KB: Extended-spectrum beta-lactamase-producing Enterobacteriaceae : an emerging public-health concern. Lancet Infect Dis 2008, 8:159–166.PubMedView Article
              6. Bonnet R: Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004, 48:1–14.PubMedView Article
              7. Humeniuk C, Arlet G, Gautier V, Grimont P, Labia R: Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. Antimicrob Agents Chemother 2002, 46:3045–3049.PubMedView Article
              8. Coque TM, Novais A, Carattoli A, Poirel L, Pitout J: Dissemination of clonally related Escherichia coli strains expressing extended-spectrum beta-lactamase CTX-M-15. Emerg Infect Dis 2008, 14:195–200.PubMedView Article
              9. Poirel L, Kampfer P, Nordmann P: Chromosome-encoded Ambler class A beta-lactamase of Kluyvera georgiana , a probable progenitor of a subgroup of CTX-M extended-spectrum beta-lactamases. Antimicrob Agents Chemother 2002, 46:4038–4040.PubMedView Article
              10. Carattoli A: Resistance plasmid families in Enterobacteriaceae . Antimicrob Agents Chemother 2009, 53:2227–2238.PubMedView Article
              11. Poirel L, Naas T, Nordmann P: Genetic support of extended-spectrum beta-lactamases. Clin Microbiol Infect 2008,14(Suppl 1):75–81.PubMedView Article
              12. Picard B, Garcia JS, Gouriou S, Duriez P, Brahimi N: The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun 1999, 67:546–553.PubMed
              13. Clermont O, Bonacorsi S, Bingen E: Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000, 66:4555–4558.PubMedView Article
              14. Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V, Demarty R, Alonso MP: Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother 2008, 61:273–281.PubMedView Article
              15. Rogers BA, Sidjabat HE, Paterson DL: Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother 2011, 66:1–14.PubMedView Article
              16. Mamlouk K, Boutiba-Ben Boubaker I, Gautier V, Vimont S, Picard B: Emergence and outbreaks of CTX-M beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol 2006, 44:4049–4056.PubMedView Article
              17. Lee MY, Ko KS, Kang CI, Chung DR, Peck KR: High prevalence of CTX-M-15-producing Klebsiella pneumoniae isolates in Asian countries: diverse clones and clonal dissemination. Int J Antimicrob Agents 2011, 38:160–163.PubMedView Article
              18. Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C: Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother 2004, 48:1249–1255.PubMedView Article
              19. Randrianirina F, Soares JL, Carod JF, Ratsima E, Thonnier V: Antimicrobial resistance among uropathogens that cause community-acquired urinary tract infections in Antananarivo, Madagascar. J Antimicrob Chemother 2007, 59:309–312.PubMedView Article
              20. Randrianirina F, Vedy S, Rakotovao D, Ramarokoto CE, Ratsitohaina H: Role of contaminated aspiration tubes in nosocomial outbreak of Klebsiella pneumoniae producing SHV-2 and CTX-M-15 extended-spectrum beta-lactamases. J Hosp Infect 2009, 72:23–29.PubMedView Article
              21. Randrianirina F, Vaillant L, Ramarokoto CE, Rakotoarijaona A, Andriamanarivo ML: Antimicrobial resistance in pathogens causing nosocomial infections in surgery and intensive care units of two hospitals in Antananarivo, Madagascar. J Infect Dev Ctries 2010, 4:74–82.PubMed
              22. Andriatahina T, Randrianirina F, Hariniana ER, Talarmin A, Raobijaona H: High prevalence of fecal carriage of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a pediatric unit in Madagascar. BMC Infect Dis 2010, 10:204.PubMedView Article
              23. Dahmen S, Bettaieb D, Mansour W, Boujaafar N, Bouallegue O: Characterization and molecular epidemiology of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in a Tunisian University Hospital. Microb Drug Resist 2010, 16:163–170.PubMedView Article
              24. Robicsek A, Jacoby GA, Hooper DC: The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis 2006, 6:629–640.PubMedView Article
              25. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D: Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006, 12:83–88.PubMedView Article
              26. Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC: Prevalence in the United States of aac(6')-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 2006, 50:3953–3955.PubMedView Article
              27. Frank T, Gautier V, Talarmin A, Bercion R, Arlet G: Characterization of sulphonamide resistance genes and class 1 integron gene cassettes in Enterobacteriaceae , Central African Republic (CAR). J Antimicrob Chemother 2007, 59:742–745.PubMedView Article
              28. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL: Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005, 63:219–228.PubMedView Article
              29. Clermont O, Dhanji H, Upton M, Gibreel T, Fox A: Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother 2009, 64:274–277.PubMedView Article
              30. Andriamanantena TS, Ratsima E, Rakotonirina HC, Randrianirina F, Ramparany L: Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar. Ann Clin Microbiol Antimicrob 2010, 9:17.PubMedView Article
              31. Pallecchi L, Bartoloni A, Fiorelli C, Mantella A, Di Maggio T: Rapid dissemination and diversity of CTX-M extended-spectrum beta-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America. Antimicrob Agents Chemother 2007, 51:2720–2725.PubMedView Article
              32. Canton R, Coque TM: The CTX-M beta-lactamase pandemic. Curr Opin Microbiol 2006, 9:466–475.PubMedView Article
              33. Herindrainy P, Randrianirina F, Ratovoson R, Ratsima Hariniana E, Buisson Y: Rectal carriage of extended-spectrum Beta-lactamase-producing gram-negative bacilli in community settings in madagascar. PLoS One 2011, 6:e22738.PubMedView Article
              34. Kim J, Kwon Y, Pai H, Kim JW, Cho DT: Survey of Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases: prevalence of SHV-12 and SHV-2a in Korea. J Clin Microbiol 1998, 36:1446–1449.PubMed
              35. Lavollay M, Mamlouk K, Frank T, Akpabie A, Burghoffer B: Clonal dissemination of a CTX-M-15 beta-lactamase-producing Escherichia coli strain in the Paris area, Tunis, and Bangui. Antimicrob Agents Chemother 2006, 50:2433–2438.PubMedView Article
              36. Novais A, Canton R, Moreira R, Peixe L, Baquero F: Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, and −32) plasmids. Antimicrob Agents Chemother 2007, 51:796–799.PubMedView Article
              37. Nuesch-Inderbinen MT, Kayser FH, Hachler H: Survey and molecular genetics of SHV beta-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrob Agents Chemother 1997, 41:943–949.PubMed
              38. Kasap M, Fashae K, Torol S, Kolayli F, Budak F: Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria. Ann Clin Microbiol Antimicrob 2010, 9:1.PubMedView Article
              39. Marcade G, Deschamps C, Boyd A, Gautier V, Picard B: Replicon typing of plasmids in Escherichia coli producing extended-spectrum beta-lactamases. J Antimicrob Chemother 2009, 63:67–71.PubMedView Article
              40. Jiang Y, Zhou Z, Qian Y, Wei Z, Yu Y: Plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in China. J Antimicrob Chemother 2008, 61:1003–1006.PubMedView Article
              41. Dahmen S, Mansour W, Boujaafar N, Arlet G, Bouallegue O: Distribution of cotrimoxazole resistance genes associated with class 1 integrons in clinical isolates of Enterobacteriaceae in a university hospital in Tunisia. Microb Drug Resist 2010, 16:43–47.PubMedView Article
              42. Chang LL, Chang TM, Chang CY: Variable gene cassette patterns of class 1 integron-associated drug-resistant Escherichia coli in Taiwan. Kaohsiung J Med Sci 2007, 23:273–280.PubMedView Article
              43. Jouini A, Ben Slama K, Vinue L, Ruiz E, Saenz Y: Detection of unrelated Escherichia coli strains harboring genes of CTX-M-15, OXA-1, and AAC(6')-Ib-cr enzymes in a Tunisian hospital and characterization of their integrons and virulence factors. J Chemother 2010, 22:318–323.PubMed
              44. Johnson JR, Stell AL, Delavari P, Murray AC, Kuskowski M: Phylogenetic and pathotypic similarities between Escherichia coli isolates from urinary tract infections in dogs and extraintestinal infections in humans. J Infect Dis 2001, 183:897–906.PubMedView Article
              45. Johnson JR, Goullet P, Picard B, Moseley SL, Roberts PL: Association of carboxylesterase B electrophoretic pattern with presence and expression of urovirulence factor determinants and antimicrobial resistance among strains of Escherichia coli that cause urosepsis. Infect Immun 1991, 59:2311–2315.PubMed
              46. Peirano G, Pitout JD: Molecular epidemiology of Escherichia coli producing CTX-M beta-lactamases: the worldwide emergence of clone ST131 O25:H4. Int J Antimicrob Agents 2011, 35:316–321.View Article

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              © Rakotonirina et al.; licensee BioMed Central Ltd. 2013

              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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