High incidence of pandrug-resistant Acinetobacter baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial.

OBJECTIVES: To investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial. METHODS: A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database. RESULTS: Of 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates. CONCLUSIONS: Carbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii.

Disparate osteogenic response of mandible and iliac crest bone marrow stromal cells to pamidronate.

OBJECTIVE: Long-term administration of intravenous bisphosphonates like pamidronate is associated with jaw osteonecrosis but axial and appendicular bones remain unaffected. Pathogenesis of bisphosphonate-associated jaw osteonecrosis may relate to skeletal site-specific effects of bisphosphonates on osteogenic differentiation of bone marrow stromal cells (BMSCs) of orofacial and axial/appendicular bones. This study evaluated and compared skeletal site-specific osteogenic response of mandible (orofacial bone) and iliac crest (axial bone) human BMSCs to pamidronate. MATERIALS AND METHODS: Mandible and iliac crest BMSCs from six normal healthy volunteers were established in culture and tested with pamidronate to evaluate and compare cell survival, osteogenic marker alkaline phosphatase, osteoclast differentiation in co-cultures with CD34+ hematopoietic stem cells, gene expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin, and in vivo bone regeneration. RESULTS: Mandible BMSCs were more susceptible to pamidronate than iliac crest BMSCs based on decreased cell survival, lower alkaline phosphatase production, and structurally less organized in vivo bone regeneration. Pamidronate promoted higher RANKL gene expression and osteoclast recruitment by mandible BMSCs. CONCLUSION: Mandible and iliac crest BMSC survival and osteogenic differentiation are disparately affected by pamidronate to favor dysregulated mandible bone homeostasis.

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme.

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii, 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606(T) and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.

Resistance to disinfectants in epidemiologically defined clinical isolates of Acinetobacter baumannii.

Decreased susceptibility to biocides may contribute to epidemic spread of Acinetobacter baumannii in the hospital. This study was conducted to evaluate the susceptibility of different clinical A. baumannii strains to disinfectants. Twenty A. baumannii strains were examined, ten of which were outbreak-related and ten that were sporadic. Clinical isolates were selected on the basis of demonstrating a unique pulsed-field gel electrophoresis pattern. The in-vitro activities of propanol, combination of 1-propanol, 2-propanol and mecetronium ethylsulphate, polyvinylpyrrolidone (PVP)-iodine, triclosan and chlorhexidine were determined using a broth macrodilution method. Exposure times to the disinfectant ranged from 15 s to 2 min and concentrations ranged from undiluted to a 1:4000 dilution in order to investigate the impact of inadvertent dilution that might occur in clinical practice. Five American Type Culture Collection (ATCC) type strains (A. baumannii, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus) were used as controls. All disinfectants inhibited growth of all isolates at concentrations and contact times recommended by the respective manufacturer. With most of the disinfectants tested, a relevant number of viable bacteria remained if contact times < 30s or diluted agents were used. No significant differences in susceptibility between outbreak-related and sporadic strains were detected, but larger studies would be required to confirm this. Resistance to currently used disinfectants is probably not a major factor in the epidemic spread of A. baumannii. However, even minor deviations from the recommended procedures leading to decreased concentrations or exposure times may play a role in nosocomial cross-transmission.

In vitro activities of isavuconazole and other antifungal agents against Candida bloodstream isolates.

Isavuconazole is the active component of the new azole antifungal agent BAL8557, which is entering phase III clinical development. This study was conducted to compare the in vitro activities of isavuconazole and five other antifungal agents against 296 Candida isolates that were recovered consecutively from blood cultures between 1995 and 2004 at a tertiary care university hospital. Microdilution testing was done in accordance with CLSI (formerly NCCLS) guideline M27-A2 in RPMI-1640 MOPS (morpholinepropanesulfonic acid) broth. The antifungal agents tested were amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and isavuconazole. C. albicans was the most common species, representing 57.1% of all isolates. There was no trend found in favor of non-Candida albicans species over time. In terms of MIC(50)s, isavuconazole was more active (0.004 mg/liter) than amphotericin B (0.5 mg/liter), itraconazole (0.008 mg/liter), voriconazole (0.03 mg/liter), flucytosine (0.125 mg/liter), and fluconazole (8 mg/liter). For isavuconazole, MIC(50)s/MIC(90)s ranged from 000.2/0.004 mg/liter for C. albicans to 0.25/0.5 mg/liter for C. glabrata. Two percent of isolates (C. glabrata and C. krusei) were resistant to fluconazole; C. albicans strains resistant to fluconazole were not detected. There were only two isolates with MICs for isavuconazole that were >0.5 mg/liter: both were C. glabrata isolates, and the MICs were 2 and 4 mg/liter, respectively. In conclusion, isavuconazole is highly active against Candida bloodstream isolates, including fluconazole-resistant strains. It was more active than itraconazole and voriconazole against C. albicans and C. glabrata and appears to be a promising agent against systemic Candida infections.

Monoclonal antibodies to vascular endothelial growth factor (VEGF) and the VEGF receptor, FLT-1, inhibit the growth of C6 glioma in a mouse xenograft.

Monoclonal antibodies raised to peptide sequences of vascular endothelial growth factor (VEGF) and the VEGF receptor, FLT-1, inhibited the growth of C6 tumors growing subcutaneously in nude mice. Immunohistochemical analysis demonstrated antibody targeting of blood vessels, tumor cells, and macrophages. A control antibody demonstrated no growth inhibition or tumor uptake. An antibody to FLT- I impaired microvascular maturation and diminished the accumulation of tumor infiltrating macrophages. The antibodies demonstrated affinity for microvasculature and tumor cells in immunohistochemistry of human glioblastoma multiforme. Targeting VEGF and its receptors has potential in the treatment of tumors of the central nervous system. FLT-1 presents an attractive target due to its presence on multiple cell types.

Cytogenetics, immunostaining for fibroblast growth factors, p53 sequencing, and clinical features of two cases of cystosarcoma phyllodes.

BACKGROUND: We present cytogenetics and fibroblast growth factor immunohistochemistry in one case of cystosarcoma phyllodes with localized disease and one with metastatic spread. The p53 gene was sequenced in the malignant case. METHODS AND RESULTS: Karyotype analysis used trypsin-Giemsa banding. Immunohistochemistry of FGF1, FGF2, FGFR1 and p53 used avidin-biotin detection of the primary antibody. One case had a mosaic female karyotype and three clones: one normal, one with trisomy 7, and one with both trisomy 5 and a rearranged chromosome 1. In the second case, a resected pulmonary metastasis had the karyotype 43-47,XX,+mar1,+mar2[6]/43-46,XX, +del(7)(p10)[3],+mar2[1][cp3]/46,XX[10]. These tumors expressed FGF1, FGF2, and FGFR1. The malignant case showed immunostaining for p53 protein, but a wild-type gene sequence. CONCLUSION: The karyotype of cystosarcoma phyllodes is complex, with wide case-to-case variation. These tumors express members of the FGF family. Metastatic behavior can occur in the presence of a wild-type p53 gene.

A four-year prospective study on microbial ecology of explanted prosthetic hips in 52 patients with "aseptic" prosthetic joint loosening.

The bacteriology of explanted prosthetic hips and surrounding soft tissue was studied in 52 patients undergoing surgical revision for joint loosening. In a prospective four-year study, positive bacterial cultures were recorded in 34 (76%) patients. Coagulase-negative staphylococci were the predominant isolates, and 11 patients (33%) had more than three organisms isolated, 7 (20%) had two only, and 11 (33%) had one species. Among the 23 patients from whom specimens from all 11 predetermined anatomic sites were cultured, the highest frequency of positive cultures (52% and 47%) came from the shaft and capsular tissue, respectively. Organisms were less frequently recovered from the cement and acetabulum (13% and 4%, respectively). Using molecular typing in eight patients with paired isolates of the same species, clonal identity was found in four. An additional patient underwent a second revision for loosening 17 months after the first revision and the same clone of Staphylococcus epidermidis was isolated on both occasions.

Methicillin-resistant Staphylococcus haemolyticus on the hands of health care workers: a route of transmission or a source?

We undertook a cross-sectional study of hand carriage and environmental contamination of methicillin-resistant coagulase-negative staphylococci on three wards of a single subspeciality surgical service. Sixteen hand cultures from 15 health care workers and 32 environmental cultures were obtained. Of 49 isolates, 35 (72%) were Staphylococcus haemolyticus. This species comprised 14 of the 16 (87%) hand isolates and 21 of the 32 (66%) environmental isolates. Using restriction length polymorphism of total DNA, we identified a single clone of S. haemolyticus on the hands of four health care workers and in the environment at seven locations on two wards. The widespread dissemination of a single clone suggests transmission of S. haemolyticus on the wards and prompts further prospective studies.

Acidic and basic fibroblast growth factors are present in glioblastoma multiforme.

Immunohistochemistry was performed on paraffin sections from human glioblastoma multiforme and normal brain tissue. Acidic fibroblast growth factor (FGF) was abundantly present in astrocytes from all glioblastomas studied. Basic FGF was found in the matrix surrounding proliferating blood vessels in most of the glioblastomas. In contrast, astrocytes from normal brain did not contain acidic FGF, and perivascular matrix staining was not demonstrated for basic FGF in the normal brain. Both growth factors could be demonstrated in neurons, Purkinje cells, capillary endothelium, and arterial walls in the normal brain. This study implicates both growth factors in the pathogenesis of malignant glioma. Both may be significant mediators of angiogenesis in glioblastoma.

Relationship between DNA strand breaks and inhibition of poly (ADP-ribosylation): enhancement of carcinogen-induced transformation.

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-amino-benzamide (3AB) for a limited period (i.e., when ADP-ribosylation is elevated) during and shortly following X-ray or MNNG-induced DNA damage of BALB/3T3 cells significantly (3- to 30-fold) enhanced transformation frequency by these agents. Individual Type III foci isolated from benzamide, X-ray, or X-ray plus benzamide treated cultures were established and characterized for growth in soft agar and for tumor induction in nude mice. DNA isolated from representative transformed lines established as a result of BA, X-ray or X-ray and BA treatments was transfected onto NIH/3T3 cells. Transformation efficiencies ranging from 0.17 to 0.28 foci/micrograms of DNA were observed suggesting the possibility that dominant transforming gene(s) were responsible for the oncogenic phenotype of radiation and benzamide transformed DNA.

Local-regional failure in patients treated with adjuvant chemotherapy for breast cancer.

Risk factors for local-regional recurrence of breast cancer were analyzed in a retrospective review of 117 patients treated with adjuvant CMF (Cytoxan [Mead Johnson & Co, Evansville, Ind], methotrexate, 5-fluorouracil) after radical or modified radical mastectomy at the Vincent T. Lombardi Comprehensive Cancer Center (Washington, DC). The median follow-up time was 50 months after mastectomy. The median time to recurrence was 23 months. The actuarial local-regional failure rate was 19% at five years. Risk of local failure correlated with size of primary (27% for T3 v 15% for T1) and axillary node status (36% for four or more positive nodes v 9% for three or fewer positive nodes). These findings suggest a rationale for the addition of postoperative radiation therapy in high-risk patients treated with adjuvant chemotherapy.