Emergence of pan-resistance in KPC-2 carbapenemase-producing Klebsiella pneumoniae in Crete, Greece: a close call.

OBJECTIVES: KPC-2-producing Klebsiella pneumoniae (KPC-KP) ST258 has been rapidly expanding and is often associated with serious nosocomial infections. Last-line antibiotics such as colistin and tigecycline often remain the only treatment option. We describe here the evolving genetic background of KPC-KP isolates in Crete, Greece. METHODS: We tested the antibiotic susceptibility of 34 clinical isolates from patients hospitalized in 2010 and 2013-14. Whole-genome sequences of these isolates were analysed for acquired resistance genes and gene mutations. RESULTS: All KPC-KP isolates belonged to ST258 with the exception of one ST147 isolate. From 2014, 26% of isolates were non-susceptible to all antibiotics, compared with 0 of 11 isolates from 2010. Colistin resistance was associated with mutations in mgrB, which was present in 61% of isolates from 2014. Core-genome MLST analysis showed that pan-resistant isolates were closely related and appeared in two separate clusters. CONCLUSIONS: KPC-KP is rapidly evolving to pan-resistance in Crete. We identified molecular resistance markers for pan-resistant isolates and showed that core-genome MLST is a promising tool for molecular fingerprinting of KPC-KP ST258.

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.

A waterborne Campylobacter jejuni outbreak on a Greek island.

A case-control and a case-crossover study were performed to investigate a Campylobacter jejuni outbreak in Crete in 2009. Most cases originated from rural areas, served by a different water-supply system from that of the adjacent town. Thirty-seven cases and 79 controls were interviewed; cases were interviewed for two different time periods for the case-crossover study. Stool cultures, PFGE and MLST subtyping were run in human samples. Univariately, consumption of tap water was associated with C. jejuni infection. Stratified analysis revealed that water-supply system was an effect modifier of this association. In the multivariable analysis, the rural areas' water supplier and drinking tap water were risk factors. No risk factors were revealed in the case-crossover study. No Campylobacter were isolated in the tested water samples. There is strong epidemiological evidence that tap water was the vehicle of the outbreak.

Synthesis and biological evaluation of trifluralin analogues as antileishmanial agents.

A series of new analogues of trifluralin (TFL) were synthesized and characterized in view of changing the unfavorable properties that limits its use as antileishmanial agent. Some of the TFL analogues display more activity than a standard drug (miltefosine) against the promastigote forms of Leishmania infantum and Leishmania donovani and the intracellular form (THP-1 infected with L. infantum). All analogues showed a clear advantage over miltefosine, as they are not hemolytic. Some analogues can conjugate these characteristics with reduced cell toxicity and improved intracellular activity.

A Chryseobacterium meningosepticum colonization outbreak in a neonatal intensive care unit.

PURPOSE: To 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 METHODS: From 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. RESULTS: Pulsed-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. CONCLUSION: This 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.

Emergence of vancomycin-resistant enterococci in a tertiary hospital in Crete, Greece: a cluster of cases and prevalence study on intestinal colonisation.

The aim of this study was to investigate the clinical and epidemiological characteristics of five consecutive cases of infection with vancomycin-resistant enterococci (VRE) and the prevalence of faecal carriage of VRE among patients admitted to a 700-bed university hospital where no VRE had been isolated previously. In a 2-month period, five consecutive patients infected with VRE were detected. Three VanB+ Enterococcus faecium isolates were obtained from three patients, while two VanA+ E. faecium isolates, one VanA+ Enterococcus faecalis isolate and one VanC1+ Enterococcus gallinarum isolate were obtained from the other two patients. Of 218 faecal specimens from all hospital wards, 41 (18.8%) were found to contain VRE. Forty-two isolates of VRE were obtained, comprising one (2%) E. faecalis, 11 (27%) E. faecium, 24 (57%) E. gallinarum and six (14%) Enterococcus casseliflavus/flavescens. Four isolates carried the vanA gene, eight carried vanB, 24 carried vanC1, and six carried vanC2/C3. Use of glycopeptides, the presence of central venous catheters and renal dialysis all correlated with VRE colonisation. The prevalence rates were among the highest reported in the literature.

Evolution of aminoglycoside resistance phenotypes of four Gram-negative bacteria: an 8-year survey in a University Hospital in Greece.

In order to determine the resistance patterns and evolution trends of four common Enterobacteriaceae (Escherichia coli, Proteus spp., Klebsiella spp. and Enterobacter spp.), aminoglycoside resistance phenotypes of 8917 non-repetitive strains, isolated over an 8-year period, were analysed. Phenotypes were defined by examining the susceptibility of the strains to a panel of aminoglycosides, using disk diffusion method. A large diversity of different resistance phenotypes was encountered. A significant progressive increase in the proportions of wild-type E. coli strains was noted. Among resistant strains of Enterobacter spp. and Klebsiella spp., the incidence of phenotype KTANt (kanamycin, tobramycin, amikacin and netilmicin), indicative of AAC(6')-I production, was very high (66.7 and 46.5%, respectively). Phenotypes indicative for gentamicin-modifying enzymes as well as broad-spectrum combinations (combinations of gentamicin-modifying enzymes with AAC(6')-I) were infrequent.

Cat scratch disease simulating a malignant process of the chest wall with coexistent osteomyelitis.

A 6-y-old girl developed fever, soft-tissue mass in the right chest wall, osteomyelitis of the 10th rib and hepatic granuloma. Cat scratch disease was diagnosed by histological examination of the mass and serological tests. The patient was treated successfully with antibiotics and recovered completely, as shown by a 10 month follow-up.

Rickettsia typhi infection in childhood.

UNLABELLED: Rickettsia typhi infection (murine typhus) is generally underdiagnosed in childhood, as clinical presentations are often non-specific. We present the manifestations in nine children hospitalized in the Department of Paediatrics of the University Hospital, Heraklion, Crete, over a 3-y period from 1998 to 2000. Titres > 1:400 for IgM and >1:960 for IgG and/or a fourfold increase in a second sample were considered strongly suggestive of acute infection. Children presented with prolonged fever, hepatosplenomegaly and lymphadenopathy. Five children presented with a rash. Unusual manifestations included aseptic meningitis and Kawasaki-like presentation. Laboratory findings included anaemia, leucopenia, and thrombocytopenia. Three children were treated with appropriate antibiotic regimens and all nine had a complete recovery. CONCLUSION: Rickettsia typhi infection should be considered in the differential diagnosis of children residing in or returning from Southern Europe countries who present with prolonged fever, rash and lymphadenopathy.

In vitro susceptibility of Coxiella burnetii to linezolid in comparison with its susceptibilities to quinolones, doxycycline, and clarithromycin.

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 microg/ml; those of doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; those of pefloxacin ranged from 1 to 4 microg/ml; and those of ciprofloxacin ranged from 4 to 8 microg/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 microg/ml.

Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA.

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 eight Leishmania 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 of Leishmania 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.

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)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.

Typing of sandflies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene.

A simple and reliable technique was developed to distinguish Phlebotomine sandflies by restriction fragment length polymorphism of PCR-amplified (PCR-RFLP) 18S rDNAs. Seven morphologically identified sandflies species from several localities of Greece and Cyprus were studied, and specific patterns were developed by double digesting amplified 18S rDNAs with HpaII and RsaI. Three additional species of the subgenus Larroussius were distinguished by a second double digestion with AccI and BanI. We have successfully applied the method on samples in which morphological characters were badly distinguished due to poor storage conditions and in larval stages.

Antimicrobial susceptibilities and beta-lactamase production of Shigella isolates in Crete, Greece, during the period 1991-1995.

The susceptibility to 11 antibiotics was determined for 52 strains of Shigella isolated from patients with diarrheal disease in Crete, Greece, during the period 1991-1995. Forty-six percent of the isolates were resistant to ampicillin, 48% to tetracycline, 44.2% to chloramphenicol, and 28.8% to cotrimoxazole. Shigella flexneri was more resistant than S. sonnei to ampicillin (82 vs 4.3%), to tetracycline (82 vs 8.7%) and to cotrimoxazole (42.8 vs 13%). Overall, 82% of all S. flexneri isolates were resistant to the three or four antimicrobial agents tested. The beta-lactamases produced by shigellae were identified by isoelectric focusing and were found to be OXA-1, TEM-1, and a low-level beta-lactamase with a pI>8. The results from the present study, which is the first carried out in Crete, emphasize the need for continuous surveillance of resistance and control of antibiotic usage.

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.

Disassembly and domain structure of the proteins in the signal-recognition particle.

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum. SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2+-depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA. The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 micrograms/ml resulted in partial loss of activity, while 10 micrograms/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.