Phenotypic and molecular characterization of macrolide and streptogramin resistance in Streptococcus mitis from neutropenic patients

OBJECTIVES: To determine the prevalence of macrolide and streptogramin resistance in Streptococcus mitis isolates from neutropenic patients and to identify mechanisms of macrolide and streptogramin resistance in resistant isolates. METHODS: MICs of erythromycin, spiramycin, lincomycin and pristinamycin were determined for S. mitis isolates. Macrolide-resistance genes were characterized by PCR and ribosomal mutations by sequencing. RESULTS: A total of 169 S. mitis isolates were recovered from 66 patients at the Tunisian Bone Marrow Transplant Centre. Of these, 120 (70%) were non-susceptible to erythromycin and one was resistant to pristinamycin; 48.5% of isolates had an MLSB phenotype with cross-resistance between erythromycin, spiramycin and lincomycin, 4% had a dissociated MLSB phenotype with resistance to erythromycin and spiramycin but apparent susceptibility to lincomycin and 47.5% displayed the M phenotype. Resistance determinants were characterized in 33 isolates. Ten of 14 isolates with the cross MLSB resistance contained an ermB-like gene and four a combination of ermB- and mefA-like genes. Four of the five isolates with a dissociated MLSB phenotype contained ermB-like and one a combination of ermB- and mefA-like genes. All the 14 isolates with an M phenotype contained mefA-like genes. The pristinamycin-resistant strain had G105 and A108 substitutions in the conserved C terminus of the L22 ribosomal protein. CONCLUSIONS: The prevalence of macrolide resistance is high in S. mitis from neutropenic patients and is due to the spread of ermB- or mefA-like genes alone or combined. Resistance to streptogramins is rare and in this case associated with ribosomal mutation.     

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.     

Isolated frontal sinus fungus ball: Diagnostic and therapeutic features and the role of endonasal endoscopic approach

Fungus ball (FB) is a non invasive form of fungal sinusitis that generally affects immunocompetent subjects. Isolated involvement of the frontal sinus is extremely rare. The treatment is surgical. Previously, it was based on the external approach. Recently, the endoscopic approach has been increasingly employed.We report three cases of frontal sinus fungus ball. Two patients underwent endoscopic endonasal frontal Draf type IIb sinusotomy with complete removal of the pathologic material. The third patient had an external approach due to the extensive pneumatisation of the frontal sinus, the defect in its floor and the orbital involvement. There were no intraoperative or postoperative complications. No recurrence of the disease was observed during the three, two and twelve months’ follow up period, respectively.Correct clinical and radiological diagnosis of isolated frontal sinus FB still remains a challenge. Endoscopic sinus surgery with endonasal Draf type IIb or type III frontal sinusotomy is effective for the treatment of frontal sinus FB. It is the treatment of choice and replaces the traditional external approaches.     

Molecular typing of Neisseria perflava clinical isolates

Multilocus sequence typing and pulsed‐field gel electrophoresis were used to type 22 commensal isolates of Neisseria perflava collected by swabbing from neutropenic patients. High genetic diversity was found among our N. perflava clinical isolates.     

First characterization in Tunisia of a TEM-15, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolate

Klebsiella pneumoniae CH0905 strain exhibiting high-level cefotaxime resistance was isolated from a stool culture in the intensive care unit. The resistance gene responsible was shown to be located on a conjugative 60-kb plasmid designated pCH0905. The minimum inhibitory concentration (MIC) values for cefotaxime and ceftazidime of the original isolate and the transconjugates were 256 mug/ml. Isoelectric focusing of a protein preparation from the K. pneumoniae strain showed beta-lactamases with the pI values of 7.6 and 6.3. A 1,080-bp fragment amplified with PCR was cloned into the pGEM-T Easy vector. The nucleotide sequence of the complete 1,080 bp was determined. Sequence analysis revealed that the bla(TEM) gene of pCH0905 differed from bla(TEM-1) by two mutations, leading to the following amino acid substitutions: the glutamic acid residue at position 104 by lysine and the glycine residue at position 238 by serine (Ambler numbering). The association of these two mutations was described previously in TEM-15 beta-lactamase, but this is the first detection of this enzyme in Tunisia.     

Variability of gB and gH genes of human herpesvirus-6 among clinical specimens

The isolates of human herpesvirus-6 (HHV-6), a betaherpesvirus closely related to human cytomegalovirus (HCMV), are classified as either variants A (HHV-6A) or B (HHV-6B) but their intravariant variability has not been studied extensively so far. The full-length genes of envelope glycoproteins gB and gH from 40 distinct HHV-6-DNA-positive specimens and 11 laboratory strains were amplified using PCR, and their nucleotide sequence determined. Nucleotide divergences were observed at 156 (6.2%) and 98 (4.7%) positions in the case of gB and gH genes respectively. Phylogenetic analysis, including reference strain sequences, confirmed the unambiguous distinction between HHV-6A and HHV-6B for both genes. In the case of HHV-6B isolates, two subgroups of gB gene (designated as gB-B1 and gB-B2) and two subgroups of gH gene (gH-B1 and gH-B2) were identified but the phylogenetic trees of both genes were not fully congruent with each other. The analysis of gB and gH protein sequences showed that 26 and 39 critical amino acid changes respectively permitted the unambiguous distinction between HHV-6A and HHV-6B. Among HHV-6B isolates, gB and gH gene subgroups were characterized by specific amino acid signatures made of six, and two residues respectively. The linkage unbalance between amino acid signatures as well as the distribution of crucial nucleotide changes strongly suggested the occurrence of intravariant recombination within gB gene among HHV-6B isolates. These results indicate that, as in the case of HCMV, homologous recombination may contribute to the genetic variability of HHV-6.     

Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity

Angiogenesis, a critical driver of tumor development, is controlled by interconnected signaling pathways. Vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 play crucial roles in the biology of normal and tumor vasculature. Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-β and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF. The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies.     

Recent advances in the research and development of RAF kinase inhibitors

The RAS-RAF-MEK-ERK signaling pathway (ERK pathway) plays a key role in tumorigenesis and cancer progression. Mutations of RAS or B-RAF lead to a constitutive activation of the ERK pathway, which ultimately results in increased cell division, and cell survival. This review article focuses on the recent literature related to ERK pathway inhibitors, with a particular emphasis on RAF kinase inhibitors. Preclinical and clinical data for the RAF kinase inhibitor sorafenib (BAY 43-9006 tosylate), that was recently approved in the US for the treatment of advanced renal cell carcinoma, are also outlined.