Ioanna Spyridaki

Detection of ciprofloxacin resistance mutations in Campylobacter jejuni gyrA by nonradioisotopic single‐strand conformation polymorphism and direct DNA sequencing

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single‐strand conformation polymorphism (non‐RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)‐amplified DNA fragments confirmed the results obtained by non‐RI SSCP analysis and revealed that in clinical strains high‐level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin ≥ 16 μg/ml] was closely associated with one type of single‐point mutation at codon 86 (Thr‐lle). Two strains with MICs of 8 and 1 μg/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala‐Thr), type II to mutation at codon 90 (Asp‐Asn), type III to variant with transition at 119, type IV to wild‐type, type V to mutation at codon 86 (Thr‐lle), and type VI to mutation at codon 86 (Thr‐lle) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non‐RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni.

First Isolation and Identification of Rickettsia conorii from Ticks Collected in the Region of Fokida in Central Greece

ABSTRACT Three different spotted-fever group rickettsiae—Rickettsia conorii, R. massiliae, and R. rhipicephali—were detected and identified by PCR-restriction fragment length polymorphism analysis in Rhipicephalus ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to R. conorii was previously demonstrated. The infection rate of ticks was 1.6%. Moreover, R. conorii was isolated from one Rhipicephalus sanguineus tick.

In Vitro Susceptibility of Coxiella burnetii to Linezolid in Comparison with Its Susceptibilities to Quinolones, Doxycycline, and Clarithromycin

ABSTRACT The in vitro susceptibility to linezolid shown by nine Greek isolates of Coxiella burnetii derived from patients with acute Q fever was investigated. MICs of linezolid were compared with those of pefloxacin, ciprofloxacin, ofloxacin, trovafloxacin, doxycycline, and clarithromycin using the shell vial assay. MICs of linezolid and clarithromycin ranged from 2 to 4 μg/ml; those of doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 μg/ml; those of pefloxacin ranged from 1 to 4 μg/ml; and those of ciprofloxacin ranged from 4 to 8 μg/ml. Linezolid was effective in controlling intracellular parasites in cultures of Vero cells infected by C. burnetii. No bactericidal activity by linezolid was obtained against C.burnetii at 8 μg/ml.

Bacteriostatic and Bactericidal Activities of Tigecycline against Coxiella burnetii and Comparison with Those of Six Other Antibiotics

ABSTRACT The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 μg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 μg/ml; linezolid and clarithromycin ranged from 2 to 4 μg/ml; and ciprofloxacin ranged from 4 to 8 μg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 μg/ml.

HA metabolism in skin homeostasis and inflammatory disease.

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities.

Colonization of Phlebotomus neglectus (Diptera: Psychodidae), the major vector of visceral leishmaniasis in Greece.

Abstract Colonization of Phlebotomus neglectus Tonnoir, the major vector of visceral leishmaniasis, in Greece is reported for the first time. Starting with wild-caught specimens, a small closed colony was established that was maintained for 17 mo or 10 generations. Gonotrophic discordance, stenogamic mating behavior, low fecundity, and dormancy because of low temperature were the most important findings that characterized the colony.

Diagnosis of quinolone‐resistant Coxiella burnetii strains by PCR‐RFLP

A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q‐fever patients, two reference strains—Nine Mile and Q212—and two pefloxacin‐resistant laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)‐amplified fragments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC)≤ 4 μg/ml] were identical. Direct DNA sequencing of the in vitro‐selected resistant strains (MICs to pefloxacin, 8–32 μg/ml) revealed a transition (G→A) at the corresponding codon 87 of E. coli. This mutation lead to the substitution of Glu (codon GAG) by Lys (codon AAG ). Restriction maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the MnlI restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR‐RFLP analysis has proved to be a simple, rapid, and useful method for the detection of Coxiella burnetii and, at the same time, for the diagnosis of quinolone‐resistant Coxiella burnetii strains. J. Clin. Lab. Anal. 14:59–63, 2000.

Role of the extracellular matrix in cancer-associated epithelial to mesenchymal transition phenomenon

The epithelial to mesenchymal transition (EMT) program is a crucial component in the processes of morphogenesis and embryonic development. The transition of epithelial to mesenchymal phenotype is associated with numerous structural and functional changes, including loss of cell polarity and tight cell–cell junctions, the acquisition of invasive abilities, and the expression of mesenchymal proteins. The switch between the two phenotypes is involved in human pathology and is crucial for cancer progression. Extracellular matrices (ECMs) are multi‐component networks that surround cells in tissues. These networks are obligatory for cell survival, growth, and differentiation as well as tissue organization. Indeed, the ECM suprastructure, in addition to its supportive role, can process and deliver a plethora of signals to cells, which ultimately regulate their behavior. Importantly, the ECM derived signals are critically involved in the process of EMT during tumorigenesis. This review discusses the multilayer interaction between the ECM and the EMT process, focusing on contributions of discrete mediators, a strategy that may identify novel potential target molecules. Developmental Dynamics 247:368–381, 2018.

Proteoglycans—Biomarkers and Targets in Cancer Therapy

Proteoglycans (PGs), important constituents of the extracellular matrix, have been associated with cancer pathogenesis. Their unique structure consisting of a protein core and glycosaminoglycan chains endowed with fine modifications constitutes these molecules as capable cellular effectors important for homeostasis and contributing to disease progression. Indeed, differential expression of PGs and their interacting proteins has been characterized as specific for disease evolvement in various cancer types. Importantly, PGs to a large extent regulate the bioavailability of hormones, growth factors, and cytokines as well as the activation of their respective receptors which regulate phenotypic diversibility, gene expression and rates of recurrence in specific tumor types. Defining and targeting these effectors on an individual patient basis offers ground for the development of newer therapeutic approaches which may act as either supportive or a substitute treatment to the standard therapy protocols. This review discusses the roles of PGs in cancer progression, developing technologies utilized for the defining of the PG “signature” in disease, and how this may facilitate the generation of tailor-made cancer strategies.