Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the ␤-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the ␤-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing ␤-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

Several expanded-spectrum ␤-lactamase bla CTX-M genes are associated with ISEcp1-like elements in Enterobacteriaceae. We found that ISEcp1B was able to mobilize the adjacent bla CTX-M-19 gene by a transpositional mechanism in Escherichia coli by recognizing a variety of DNA sequences as right inverted repeats.

ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum ␤-lactamase bla CTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the bla CTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the bla CTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 g/ml). In those cases, ISEcp1B brought promoter sequences enhancing bla CTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the bla CTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-bla CTX-M-2 occurred at (6.4 ؎ 0.5) ؋ 10 ؊7 in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40°C.

Genes encoding extended-spectrum ␤-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla CTX-M-1 gene located on transferable plasmids of different sizes and structures.

Corynebacterium ulcerans causes zoonotic infections, such as diphtheria and extrapharyngeal infections. We report here the first case of a diphtheria-like illness caused by C. ulcerans in France and transmitted likely by a dog to an immunocompromised woman. CASE REPORTA 47-year-old woman was admitted in the emergency room at the Bicêtre University Hospital (Le Kremlin-Bicêtre, France) for severe dyspnea in October 2003. She was immunocompromised due to treatment by prednisone at 6 mg/daily, tacrolimus at 12 mg/daily, and mycophenolate nofetil at 1 g/daily since she had undergone a kidney graft 1 year previously. Ten days prior to her admission to Bicêtre Hospital, the patient had been treated to no avail for sinusitis by oral amoxicillin-clavulanic acid (2 g/daily).Physical examination revealed severe stridor. She had fever at 38°C and mild tachycardia. The endoscopic examination identified a pseudomembranous exudate covering the nasopharynx and laryngeal vestibulum, ulceration, and subglottic constriction. A blood test found an increase of C-reactive protein (100 g/ml) and a white blood cell count of 3,500/l. This severe dyspnea indicated intubation, and the patient was subsequently hospitalized in the intensive care unit.Throat swab showed coryneform and gram-positive rods. After 24 h of incubation on sheep blood agar at 37°C in 5% CO 2 , shiny and whitish colonies grew that produced a slight hemolysis. Microscopic examination of these colonies revealed gram-positive, coryneform rods arranged in palisades. They were catalase positive, showed a positive reaction for urease, were negative for pyrazinamidase, and fermented glucose, ribose, maltose, and glycogen. These characteristics corresponded to those of Corynebacterium ulcerans. Biochemical identification was confirmed by partial sequencing of the 16S rRNA gene that showed 99% identity of the sequence of that strain with that of a C. ulcerans reference strain (11).Since C. ulcerans may acquire lysogenic ␤-corynephages coding for a diphtheria-like toxin (DT), PCR test (using primers DT1 and DT2 for detection of tox gene of C. diphtheriae (6) was performed that showed the presence of the DT-like gene in C. ulcerans. Disk diffusion susceptibility testing was performed on Mueller-Hinton blood agar (supplemented with 5% [vol/vol] sheep blood) after overnight incubation at 35°C and 5% CO 2 . In the absence of standardized breakpoints for Corynebacterium, antibiotic susceptibility was determined by using the NCCLS criteria for Streptococcus spp. other than Streptococcus pneumoniae (10).

Variations in proteins related to bacterial diversity may affect species identification performed using matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Using this method, we identified 110 Streptococcus agalactiae isolates characterized by serotyping and multilocus sequence typing. Serotype III and sequence type 23 strains expressed the widest variation in molecular weight of putative "species-identifying" biomarker ions. Recognition of the diversity of MALDI patterns observed in strains that represent all major intraspecies lineages assists in the constitution of an optimal reference database.

BackgroundStreptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing.ResultsOur study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively.ConclusionsThis comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1583-4) contains supplementary material, which is available to authorized users.

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