A total of 922 non-duplicate isolates (one per patient) of Enterobacteriaceae were collected from March 2005 to April 2010 from the Microbiology Laboratories of five hospitals and four private laboratories in Bejaia (Algeria). The isolates were recovered from various pathological specimens and were identified by the API 20E system (bioMérieux, Marcy l’Etoile, France) as follows: E. coli (n=551); Klebsiella pneumoniae (n=221); P. mirabilis (n=125) and Salmonella sp. (n=25).

E. coli J53AzR was used as recipient strains for conjugation experiments. E. coli DH10B (Invitrogen) was used in transformation experiments and E. coli ATCC 25922 was used as a quality control strain for antimicrobial susceptibility testing.

Antibiotic susceptibility was determined on Mueller Hinton agar by standard disk diffusion procedure as described by the European Committee on Antimicrobial Susceptibility Testing (2014),13 for the following antibiotics: aztreonam, ticarcillin, piperacillin, amoxicillin–clavulanate, ticarcillin–clavulanate, cefoxitin, cefepime, piperacillin–tazobactam, cefuroxime, cefotaxime, ceftazidime, imipenem, tobramycin, amikacin, gentamicin, sulfonamide, trimethoprim, nalidixic acid, ciprofloxacin, norfloxacin, tetracycline, and chloramphenicol (BioRad, Marnes La Coquette, France). For tetracycline, the Antibiogram Committee of the French Society for Microbiology recommendations breakpoints were used (http://www.sfm-microbiologie.org).

Isolates showing a zone of inhibition diameter ≤20mm with cefoxitin were selected for screening for pampC genes. ESBL production was detected by a double-disk synergy test (DDST) on Mueller Hinton supplemented with cloxacillin (200mg/L).14 Inducibility of the β-lactamase was determined by the double disk test. The cephalosporins used were cefotaxime, ceftazidime, and cefepime. Clavulanic acid (10μg) and cefoxitin (30μg) were used as inducing agents. The plates were examined after overnight incubation at 37°C.15

Minimum inhibitory concentrations (MICs) of amoxicillin, amoxicillin/clavulanate, piperacillin/tazobactam, cefotaxime, ceftazidime, cefoxitin, imipenem, aztreonam, and cefepime were determined by Etest (AB bioMérieux, Marcy l’Etoile, France).

Total DNA was extracted by using a QIAmp DNA Mini Kit (QIAGEN) according to the instructions of the manufacturer. A multiplex PCR covering the six families of ampC genes (CMY-2/BIL/LAT, CMY-1/MOX, DHA, FOX, ACC, ACT/MIR) was performed as previously described.16 PCR-positive isolates were further tested using individual pairs of primers and then sequenced. pAmpC-producing isolates positive for the DDST were screened for blaCTX-M, blaTEM and blaSHV by PCR as described previously.17

Screening of qnrA, qnrB, qnrS, qnrC, qnrD and qepA genes was carried out with a multiplex real-time PCR assay using SYBR Green I and Roche LightCycler1 as described previously.18 Pyrosequencing method was used for the detection of aac(6′)-Ib-cr and aac(6′)-Ib genes.19

All PCR products were sequenced and the sequencing results were compared to reported sequences available in GenBank.

Conjugation was performed on Mueller Hinton agar supplemented with sodium azide (100mg/L) and cefotaxime (1mg/L). Transconjugants growing on the selection plates were subjected to antimicrobials susceptibility, DDST and PCR analysis to confirm the presence of the AmpC phenotype.

Possible genomic relatedness of strains was analyzed by RAPD using genomic DNA as previously described.20 Multilocus sequence typing (MLST) was performed on the K. pneumoniae isolates using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB).21 A detailed protocol of the MLST procedure, including allelic type and ST assignment methods, is available in MLST databases from the Pasteur Institute, Paris, France, at the website http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html.

PCRs were performed to determine the phylogenetic groups (A, B1, B2, C, D, E, F, and clade I) of the E. coli isolates, using the newly revised Clermont method.22 All isolates belonging to group B2 were analyzed by two multiplex PCR as described previously.23 The presence of eight virulence factors found in ExPEC was investigated by PCR. These factors included sfa/foc (S and F1C fimbriae), papG alleles (G adhesin classes of P fimbriae), papC (C adhesin classes of P fimbriae), hlyA (alpha-haemolysin A), cnf (cytotoxic necrotizating factor 1), fyuA (genes of yersiniabactin), iutA (aerobactin receptor), and ibeA (invasion protein IbeA).

Plasmids incompatibility (Inc) groups were determined using PCR-based replicon typing (PBRT).24 Four multiplex PCR were used for the detection of A/C, T, FIIAs, W, N, FIB, L/M, I1-Iγ, X, HI2, FIA, and Y replicons. Replicons P, R, U, F, FIC, HI1, B/O and K were detected by simplex PCR.24,25 Replicons FII1K, FII2K, NewXXX also named ZK, LVPK, and Amet were detected using PCR method described by D. Decré and G. Arlet.

For the analysis of genetic arrangement of the resistance genes, overlapping PCR amplification of internal regions of the transposon-like element that carried blaCMY-4 was performed based on known sequences.

Genetic structures surrounding the blaDHA-1 gene were studied by PCR mapping, cloning and sequencing method using a large variety of primers based on the previously reported structures.8

The nucleotide sequence and the deduced protein sequence were analyzed using the Basic Local Alignment Search Tool (BLAST) through the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignment of deduced peptide sequences was carried out with the Vector NTI program (Invitrogen).

Among the 922 Enterobacteriaceae isolates, 15 isolates showed decreased susceptibility to cefoxitin: nine isolates of E. coli (9/551), five isolates (5/221) of K. pneumoniae and one isolate (1/125) of P. mirabilis. Ten isolates were recovered from urine. Six E. coli isolates and one K. pneumoniae isolate were from community patients (7/712), and the remaining eight isolates were collected from hospitalized patients (8/210).

All isolates exhibited resistance to ticarcillin, piperacillin, ticarcillin–clavulanic acid, amoxicillin–clavulanic acid, cefuroxime, cefotaxime, ceftazidime, aztreonam, and cefoxitin. Isolates exhibited intermediate resistance to piperacillin–tazobactam (60%) and cefepime (40%). Resistance of the isolates to non-β-lactam antibiotics was high for sulfonamide (80%), tobramycin (71.4%), gentamicin (71.4%), tetracycline (71.4%), chloramphenicol (64.3%) and trimethoprim (57.1%), and low for nalidixic acid (28.6%) and amikacin (6.6%). The isolates remain susceptible to imipenem and fluoroquinolones. The MICs ranges are listed in Table 1.

The ESBL phenotypic screening by double disk diffusion synergy test showed that one isolate of K. pneumoniae and two isolates of E. coli were ESBL producers (Table 1).

Inducibility of β-lactamases was recognized by the disk antagonism test, which demonstrated blunting of the cephalosporin disks adjacent to the cefoxitin and clavulanic acid disks in only one isolate of K. pneumoniae 413. This phenotype suggested the presence of an inducible AmpC-type β-lactamase.

By multiplex PCR, we obtained amplicons in 15 isolates: nine E. coli isolates, five K. pneumoniae isolates, and one P. mirabilis isolate (Table 1).

PCR and sequencing analysis revealed the presence of blaCMY-4 in all isolates except one isolate of K. pneumonia, which produced blaDHA-1.

In addition, two E. coli (CMY-4) co-produced CTX-M-15 ESBLs and one isolate of K. pneumoniae (DHA-1) co-produced CTX-M-3 and SHV-12 ESBL. All isolates carried blaTEM-1.

PCR amplification of PMQR yielded amplification in one K. pneumoniae isolate only. This strain expressed both the qnrB4, aac(6′)-Ib, blaDHA-1, blaCTX-M-3, blaSHV-12 and blaTEM-1 genes (Table 1).

No amplicons were obtained for qnrA, qnrS, qnrD, qnrC and qepA in all isolates.

By mating assay, the ampC genes were transferred from three of the five K. pneumoniae, five of the nine E. coli isolates and from the P. mirabilis isolate. Susceptibility results of the transconjugants are shown in Table 1.

PBRT of the plasmid Inc groups showed that the plasmids carrying blaCMY-4 belonged to the Inc A/C group and the plasmid carrying blaDHA-1 belonged to the group Inc LVPK (Table 1).

RAPD-typing revealed the presence of diverse bacterial population and no predominant clone was identified in our collection.

MLST analysis of the five AmpC-producing K. pneumoniae identified four different STs, including ST17, ST834 and two new sequence types: ST1617 (Kp 123) and ST1618 (Kp 613 and 615) (Table 1). In ST1618, we described a new allele's mdh and rpoB designated respectively 145 and 108. The typing results generated by RAPD analysis among the isolates were compatible with those obtained by MLST.

Of the nine E. coli isolates, three belonged to group D, three to group B1 (recovered from urine), two to group B2, and the last one to group F (Table 2).

Five isolates harbored genes encoding siderophores (fyuA, iutA).

The E. coli 412 isolate was assigned to the B2 sub-group VII and STc14. This strain was isolated from the feces of a hospitalized patient. This isolate contained a blaTEM-1 gene and, a blaCMY-4 gene, which was transferred with an Inc A/C plasmid. The following virulence genes (papC, papG II, sfa, hlyA, cnf1, fyuA and iutA) were detected in this isolate (Table 2).

By using allele-specific PCR method for detecting the main E. coli B2 STc, E. coli 611 isolate was unassigned; it did not give any PCR products except for the internal control.

Analysis of the genetic structure of the blaCMY-4 gene in our collection showed that it was located on a transposon-like DNA element consisting of a specific ISEcp1/ΔISEcp1-blaCMY-4-blc-sugE structure. This structure was similar to that found in plasmid pCC416 (GenBank AJ875405).

A region typical of a complex sul1-type integron, from the int gene to CR1 was amplified using PCR mapping and then sequenced. By cloning the region encompassing ampC and ampR, a recombinant plasmid (p413C) with an insert that conferred inducible resistance to ceftazidime was selected. The insert was found to contain blaDHA-1 and the regulatory gene ampR, which was downstream of blaDHA-1. This insert shared also part of pRBDHA's backbone carrying a complex integron (GenBank AJ971343). PCR and DNA sequencing results confirmed that the plasmid encoded at least three β-lactamase genes: blaTEM-1, blaSHV-12, and blaDHA-1, and a plasmid mediated resistance to quinolone (QnrB4).