Efstathia Scoulica

Detection and Identification of Leishmania DNA within Naturally Infected Sand Flies by Seminested PCR on Minicircle Kinetoplastic DNA

ABSTRACT A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eightLeishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles ofLeishmania spp. in areas of endemicity.

Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA

Relationships among seventy specimens, fifteen species and three genera of phlebotomines were inferred from the phylogenetic analysis of small subunit nuclear rDNA, obtained by the PCR amplification and cloning of almost full‐length genes. Outgroups included fifteen dipterans, and single representatives of four other insect orders. The more distant the taxa compared, the larger were the regions of ambiguous sequence alignment that needed to be deleted in order to avoid circularity in performing parsimony analyses. Phlebotomine sequences formed a monophyletic clade within the suborder Nematocera, with the progressively more basal sister groups of Diptera being Culicomorpha, Tipulomorpha and the suborder Brachycera. Within Phlebotominae, subgeneric relationships were resolved and the genus Phlebotomus was shown to be monophyletic, but markers for intraspecific geographical populations were not found and intergeneric relationships were not resolved.

Simultaneous Detection of “Rickettsia mongolotimonae” in a Patient and in a Tick in Greece

ABSTRACT Rickettsia conorii, a spotted fever group rickettsia which is transmitted by Rhipicephalus sp. complex ticks, was considered until now the only pathogenic rickettsia prevalent in Greece. Here, we report the presence of “Rickettsia mongolotimonae” (proposed name) detected simultaneously in a patient and in a Hyalomma anatolicum excavatum tick, sampled on the patient.

A Phage Tail-Derived Element with Wide Distribution among Both Prokaryotic Domains: A Comparative Genomic and Phylogenetic Study

Prophage sequences became an integral part of bacterial genomes as a consequence of coevolution, encoding fitness or virulence factors. Such roles have been attributed to phage-derived elements identified in several Gram-negative species: The type VI secretion system (T6SS), the R- and F-type pyocins, and the newly discovered Serratia entomophila antifeeding prophage (Afp), and the Photorhabdus luminescens virulence cassette (PVC). In this study, we provide evidence that remarkably conserved gene clusters, homologous to Afp/PVC, are not restricted to Gram-negative bacteria but are widespread throughout all prokaryotes including the Archaea. Even though they are phylogenetically closer to pyocins, they share key characteristics in common with the T6SS, such as the use of a chaperon-type AAA+ ATPase and the lack of a host cell lysis mechanism. We thus suggest that Afp/PVC-like elements could be classified as phage-like-protein-translocation structures (PLTSs) rather than as pyocins. The reconstruction of phylogeny and the conserved gene content suggest that the diversification of prophage sequences to PLTS occurred in bacteria early in evolution and only once, but PLTS clusters have been horizontally transferred to some of the bacterial lineages and to the Archaea. The adaptation of this element in such a wide host range is suggestive of its versatile use in prokaryotes.

A Chryseobacterium meningosepticum colonization outbreak in a neonatal intensive care unit

PurposeTo report the epidemiologic, bacteriologic, and clinical features of a Chryseobacterium meningosepticum outbreak in a neonatal intensive care unit (NICU) of a referral teaching hospital.Patients and methodsFrom April to October 2002, a strain of C. meningosepticum was isolated from four neonates in the NICU. All neonates were colonized in the endotracheal tubes and respiratory secretions, but none of them progressed to clinical infection. Multiple samples were obtained for cultures.ResultsPulsed-field gel electrophoresis (PFGE) of isolates showed them to be representatives of a single strain. Environmental surveillance did not reveal the C. meningosepticum source. None of the neonates received specific treatment. The outbreak was only controlled by reinforcement of the usual measures and no additional colonization/infection was confirmed for more than a year after the last case.ConclusionThis study suggests that C. meningosepticum colonization in neonates does not necessarily lead to infection and that such colonization outbreaks may be controlled with emphasis on the standard precautions.

Molecular characterization of the OXA-7 beta-lactamase gene.

The OXA-7 gene, which encodes an oxacillinase, was cloned from plasmid pMG202 of Escherichia coli isolate 7181 (A. A. Medeiros, M. Cohenford, and G. A. Jacoby, Antimicrob. Agents Chemother. 27:715-719, 1985) and sequenced. The nucleotide sequence of the OXA-7 gene was closely related to that of the OXA-10 (PSE-2) gene, with a derived amino acid sequence of the OXA-7 enzyme showing greater than 95% homology with those of OXA-10 and OXA-11.

Infant colonization by Staphylococcus aureus: role of maternal carriage

Infant colonization by Staphylococcus aureus has not been adequately investigated. In this study, we aimed to define determinants associated with the carriage of S. aureus in early infancy. Serial nasal swabs were collected from 128 infants and their mothers at months 0, 6, and 12 postpartum. S. aureus isolates were characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, and the presence of chromosomal mecA and of Panton–Valentine leukocidin (PVL) genes. S. aureus was isolated in 17.7% and 15.7% of swabs from infants and mothers, respectively. Carriage rates were higher in infants with carrier mothers, non-smoking mothers, and many siblings. Persistent carriage rates were higher in infants with carrier or non-smoking mothers. S. aureus typing revealed identical strains in 10/15 investigated infant–mother pairs. Among 19 investigated S. aureus isolates from infants, ten harbored mecA and two harbored PVL genes, and these determinants were concomitantly present in isolates from mothers. Resistance to methicillin was 43.6% among all isolates from infants. In conclusion, isolates from infants were commonly identical to isolates from their mothers, pointing to a principal role of maternal carriage in S. aureus colonization in infants.

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.

Molecular Epidemiology of Extended-Spectrum β-Lactamases Produced by Clinical Isolates in a University Hospital in Greece: Detection of SHV-5 in Pseudomonas aeruginosa and Prevalence of SHV-12

To assess the nature and diversity of various types of SHV and TEM derivatives in our hospital a survey was conducted. Sixty-seven extended-spectrum beta-lactamases (ESBL)-producing nosocomial pathogens, isolated over a 12-month period, were analyzed by means of PCR and direct sequencing. SHV-5 was the predominant ESBL found in our region (38 strains). Other less frequent variants included SHV-2 and SHV-12 with two and three isolates, respectively. For the first time, an outbreak of 11 Pseudomonas aeruginosa producing SHV-5 was encountered. All blaTEM-positive strains carried the non-ESBL TEM-1. The incidence of non-SHV non-TEM ESBLs was remarkably high as almost one out of three isolates harbored such an ESBL. The epidemiological and clinical impact of these findings must be carefully investigated and interpreted.

First isolation and genotypic identification of Rickettsia conorii Malish 7 from a patient in Greece

Described here is the first isolation of Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), from a patient in Greece. Molecular characterization identified the strain as R. conorii Malish 7. Although R. conorii has previously been recovered from ticks in this country, this is the first time the pathogen has been recovered from a human. On 6 December 2002, a 39-year-old male resident of Sitia (a small town in a rural area of eastern Crete, Greece), was admitted to the Internal Medicine Department of the General Hospital of Sitia with fever (up to 39.8◦C), chills, arthralgia of the arm and leg joints, headache and cough of 5 days’ duration. Two days prior to admission he had noticed the presence of a skin lesion on his right tibia. Physical examination at admission revealed the patient to be febrile (39◦C) with headache and arthralgia of the knee. An eschar (tâche noire) of 0.5 cm in diameter surrounded by an inflammatory halo (2–2.5 cm in diameter) was noted on the outer surface of the distal end of the right tibia. A hyperemic maculopapular rash was also noted on the patient’s trunk and extremities. The rash was primarily concentrated on the sides of the trunk and on the patient’s shoulders, but its was also present on the palms of the hands and on the sides of the soles of the feet. The elements of the rash were 0.3–0.7 mm in diameter, and some had a central blister with a size comparable to that of a pinpoint. No petechia was found. Clinical examination was otherwise normal. Laboratory findings included transient leukocytosis (13,200/mm3 on day 1, and 5,500/mm3 on day 2), a moderately elevated erythrocyte sedimentation rate of 58 on day 1,