Fluoroquinolone Resistance Mechanisms and population structure of Enterobacter cloacae non-susceptible to Ertapenem in North-Eastern France.

Fluoroquinolone (FQ) agents are a potential resort to treat infection due to Enterobacteriaceae producing extended spectrum ß-lactamase and susceptible to FQ. In a context of increase of non-susceptibility to carbapenems among Enterobacteriaceae, we characterized FQ resistance mechanisms in 75 Enterobacter cloacae isolates non-susceptible to ertapenem in North-Eastern France in 2012 and describe the population structure by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Among them, 14.7% (12/75) carried a carbapenemase-encoding gene. Except one isolate producing VIM-1, the carbapenemase-producing isolates carried the well-known IncL/M pOXA48a plasmid. Most of the isolates (59/75) harbored at least a FQ-R determinant. qnr genes were predominant (40%, 30/75). The MLST study revealed that E. cloacae isolates' clonality was wide [24 different sequence types (STs)]. The more widespread STs were ST74, ST101, ST110, ST114, and ST133. Carbapenem MICs were higher for E. cloacae ST74 than for other E. cloacae isolates. Plasmid-mediated quinolone resistance determinants were more often observed in E. cloacae ST74 isolates. These findings showed that (i) pOXA-48a is spreading in North-Eastern France, (ii) qnr is preponderant in E. cloacae, (iii) E. cloacae comprised a large amount of lineages spreading in North-Eastern France, and (iv) FQ as an alternative to ß-lactams to treat ertapenem non-susceptible Enterobacteriaceae are compromised.

Mobile insertion cassette elements found in small non-transmissible plasmids in Proteeae may explain qnrD mobilization.

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36-60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae.

A simplified and cost-effective method combining real-time PCR and pyrosequencing for detection of aac(6')-Ib-cr gene.

We previously described a pyrosequencing-based method able to guarantee detection of aac(6')-Ib-cr by characterizing precisely the single nucleotide polymorphisms leading to Trp102Arg and Asp179Tyr. By gaining in simplicity and cost-effectiveness, through the use of real-time PCR, we propose here a cutting-edge evolution of our existing pyrosequencing method.

First report of qnrB-producing Enterobacter cloacae and qnrA-producing Acinetobacter baumannii recovered from Algerian hospitals.

Two isolates of Enterobacter cloacae and 1 isolate of Acinetobacter baumannii showing a multidrug resistance phenotype including resistance to beta-lactams and fluoroquinolones were included in this study. By polymerase chain reaction amplification and sequencing, the 2 isolates of E. cloacae were found to produce QnrB and the A. baumannii isolate was found to produce QnrA. In addition, the 2 E. cloacae isolates were found to produce CTX-M-15.

Characterization of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from hospital environments in Algeria.

For detecting extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the hospital environment, sedimentation plates were placed in the rooms of two hospitals. Three environmental isolates, two Klebsiella pneumoniae, and one Escherichia coli resistant to extended-spectrum cephalosporins with a phenotype indicating CTX-M enzymes production (the minimum inhibitory concentration [MIC] of cefotaxime was higher than the MIC of ceftazidime) were recovered. By PCR and sequencing, the three isolates were found to produce CTX-M-15. The bla(CTX-M-15) genes in the three isolates were transferred by conjugation. One K. pneumoniae environmental isolate showed an identical and unique RAPD profile with two other K. pneumoniae clinical isolates recovered from urinary tract infection from patients hospitalized in two different wards of another hospital.

First report of CTX-M-15 and CTX-M-3 beta-lactamases among clinical isolates of Enterobacteriaceae in Béjaia, Algeria.

We assessed the prevalence and phenotypic characteristics of extended-spectrum beta-lactamase producers among 365 clinical isolates of members of the family Enterobacteriaceae recovered from two hospitals in Béjaia, Algeria, between March 2004 and April 2005. Twenty-one strains were resistant to cefotaxime and/or ceftazidime. A double-disk synergy test yielded a positive result in five cases (three Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae). Using polymerase chain reaction and sequencing, the three E. coli isolates and the K. pneumoniae isolate were found to produce extended-spectrum beta-lactamase CTX-M-15 and the E. cloacae isolate produced CTX-M-3. The three CTX-M-15-producing E. coli isolates were not isolated in the same wards, although genotyping by randomly amplified polymorphic DNA analysis revealed that they were clonally related. The bla(CTX-M-15) genes were transferred from E. coli by conjugation, whilst conjugative transfer of bla(CTX-M) genes from K. pneumoniae and E. cloacae was not detectable.