Parstatin inhibits viability of human retinal pigment epithelium cells: an in vitro cytotoxicity study.

OBJECTIVE: Parstatin, the N-terminal 41-amino-acid peptide cleaved by thrombin from protease-activated-receptor 1, was shown to be highly effective in preventing ocular angiogenesis and as such it has potential therapeutic applications in ocular neovascular diseases. In the frame of a safety program in preclinical development, we investigated whether parstatin exerts any cytotoxic effect in critical ocular cell populations. MATERIALS AND METHODS: Human retinal pigment epithelium cell-19 line (ARPE-19) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay were used to investigate parstatin's effect in cell cultures. Parstatin 24-41 was used as a control peptide which lacks the hydrophobic domain to demonstrate the specificity and the structure-dependent effect of parstatin. Both peptides were used at concentrations ranging from 0.1-30 µM for 24, 48 and 72 hours. RESULTS: In the presence of parstatin the rate of ARPE-19 cell growth and viability were significantly decreased in a concentration-dependent and time-dependent manner, with an IC50 of approximately 10 µM. When ARPE-19 cells were treated with parstatin 24-41 no inhibitory effect was observed at any concentration or exposure time used. CONCLUSIONS: Parstatin has a clear detrimental effect on the viability of ARPE-19 cells and raises concerns about its use in the eye because of its possible toxic effects.

Postpartum bacteraemia outbreak due to Bacillus cereus in the delivery room.

We report an outbreak Bacillus cereus causing postpartum bacteraemia in the maternity ward and delivery room. Spores transferred by the hands and gloves of the staff in the maternity ward contaminated equipment in these two areas.

Carbapenemase-producing Klebsiella pneumoniae bloodstream infection in critically ill patients: risk factors and predictors of mortality.

A significant increase in carbapenemase-producing Klebsiella pneumoniae (CP-Kp) bacteraemias has been observed worldwide. The objective of the present work was to study the risk factors and predictors of mortality of CP-Kp bacteraemias among critically ill patients. During a 4-year period (2012-3015), a matched 1:2 case-control study was conducted. Klebsiella pneumoniae was identified by Vitek 2 technology. Antibiotic susceptibility was performed by the agar disc diffusion method and Etest. The presence of the bla KPC, bla VIM and bla NDM genes was confirmed by polymerase chain reaction (PCR). Epidemiologic data were collected from the intensive care unit (ICU) computerised database. One hundred and thirty-nine patients who developed a CP-Kp bacteraemia were matched with 278 patients. The majority of isolates (128; 92.1%) carried the bla KPC gene, seven carried both bla KPC and bla VIM, three bla VIM and one carried bla NDM. Risk factors for the development of CP-Kp bacteraemia were administration of tigecycline and number of antibiotics administered prior to CP-Kp bacteraemia. Overall, the 30-day mortality was 36.0%. Multivariate analysis revealed septic shock, Simplified Acute Physiology Score II (SAPS II) upon infection onset, adjunctive corticosteroid administration and parenteral nutrition as independent predictors of mortality, while treatment with a combination of appropriate antibiotics was identified as a predictor of good prognosis. Among septic shock patients (n = 74), Sequential Organ Failure Assessment (SOFA) score upon infection onset, adjunctive corticosteroid administration and strain carrying the bla KPC gene were independently associated with mortality, while the administration of combination treatment was identified as a predictor of a good prognosis. The administration of tigecycline predisposes to the induction of bacteraemia. Appropriate antibiotic treatment is associated with better survival, while concomitant corticosteroid treatment is associated with mortality.

Pitfalls in the identification of Enterococcus species and the detection of vanA and vanB genes.

UNLABELLED: The aims were to assess the performance of Vitek 2 in identifying enterococcal species and the implementation of GeneXpert(®) vanA/vanB PCR for the detection of vancomycin-resistant enterococci (VRE). Gram-positive cocci from clinical and environmental specimens (n = 431) suspicious of being enterococci by conventional methods were evaluated by Vitek 2. This system identified 296 Enterococcus faecium, 87 Enterococcus faecalis, 10 Enterococcus villorum, 9 Enterococcus gallinarum, 9 Enterococcus durans, 5 Enterococcus casseliflavus, 1 Enterococcus spp. and 14 isolates as Non-Enterococcus. All strains were submitted to pulsed field gel electrophoresis (PFGE) analysis showing 64 banding patterns. Representative strains from each banding pattern were further characterized to species level by 16S rDNA sequencing. The misidentification rate by Vitek 2 to species level among 429 molecularly identified enterococci was 6% (26 isolates). Additionally, 372 rectal swabs were obtained from critically ill patients. They were evaluated for the presence of VRE by ChromID VRE combined with in-house PCR vs GeneXpert(®) . GeneXpert(®) showed high (>92%) sensitivity, specificity, accuracy for vanA-positive Enterococcus detection, as well as, sensitivity and specificity for vanB-positive strains. Positive predictive value for detection of vanB-positive enterococci by GeneXpert(®) vanA/vanB was low (30%). GeneXpert(®) showed the same efficacy as ChromID VRE in detecting vanA-positive enterococci, but lower for vanB-gene detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that even though the performance of Vitek 2 Advanced Expert System was good in identifying enterococci to species level, it is important to verify results by a molecular method when phenotypic findings are discordant with epidemiologic patterns. Furthermore, GeneXpert(®) vanA/vanB PCR and ChromID VRE combined with in-house PCR were applied in rectal samples for the detection of VRE colonization among critically ill patients. GeneXpert(®) showed an excellent performance in detecting vanA-positive enterococci, but false-positive results for vanB-gene detection render its application problematic in departments with high incidence of vanB-positive enterococci.

Number of positive blood cultures, biofilm formation, and adhesin genes in differentiating true coagulase-negative staphylococci bacteremia from contamination.

The significance of the number of coagulase-negative staphylococci (CNS)-positive blood cultures remains obscure in regards to determining true bacteremia versus contamination. The goal of this study was to determine the predictors of real CNS bloodstream infection among intensive care unit (ICU) patients. ICU patients with at least one CNS-positive blood culture were identified from the microbiology database. Biofilm formation was tested by glass tube and microtiter plate assay. mecA gene, ica operon genes (icaA, icaB, icaD), and adhesin genes (aap, bap, atlE, fbe, fnbA) were detected by polymerase chain reaction (PCR). CNS were recovered from 120 septic episodes, 20 of which were true CNS bacteremias, whereas from the remaining 100 episodes, the isolated CNS were characterized as contaminants. The number of positive blood cultures was significantly associated with true CNS bacteremia. Nineteen true bacteremic Staphylococcus epidermidis strains were compared to 38 contaminants. Biofilm synthesis was documented in 37 isolates associated with the presence of the ica operon (p = 0.048). There were 39, 26, 38, 21, and 10 strains positive for the presence of atlE, bap, fbe, aap, and fnbA genes, respectively. Rifampicin resistance, absence of severe sepsis, number of S. epidermidis-positive blood cultures, and absence of the bap gene were independently associated with true S. epidermidis bacteremia as compared to contaminant strains. The number of positive blood cultures is associated with true CNS bacteremia. The presence of adhesin genes may play a role in differentiating true infection from contamination, whereas absence of the bap gene is associated with true S. epidermidis bacteremia.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece.

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst-negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred.

Co-colonization by multidrug-resistant bacteria in two Greek intensive care units.

Our goal was to identify the risk factors for co-colonization by KPC-producing Klebsiella pneumoniae (KPC-Kp), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) upon intensive care unit (ICU) admission and during stay. Rectal and nasal samples were taken from each patient upon admission at two Greek ICUs and each week afterwards, and were inoculated onto chromogenic agar. Representative colonies were characterized with standard methods and Vitek-2 technology. The presence of the bla KPC gene (K. pneumoniae isolates), vanA and vanB (Enterococcus faecium and E. faecalis isolates), and mecA (S. aureus isolates) was confirmed by polymerase chain reaction (PCR). Upon ICU admission, among 481 patients, 59 (12%), 63 (13%), and 20 (4%) were colonized by KPC-Kp, VRE, or MRSA, respectively. Simultaneous colonization by KPC-Kp and VRE upon admission (34 patients) was associated with the number of co-morbidities [adjusted odds ratio (aOR): 1.5; confidence interval (CI) 1.0-2.5], administered antibiotics (aOR: 1.7; CI 1.3-2.3), and prior KPC-Kp infection (aOR: 24.4; CI 1.5-396.0). Among patients with an ICU stay of more than 6 days, 181 (73%), 31 (13%), and 9 (4%) became KPC-Kp, VRE, or MRSA colonized during ICU stay, respectively. KPC-Kp colonization was an independent risk factor for VRE colonization upon admission (aOR: 2.7; CI 1.0-7.2) and during stay (aOR: 7.4; CI 2.0-27.4), whereas VRE colonization was a risk factor for KPC-Kp upon admission (aOR: 5.1; CI 1.9-13.9) and MRSA colonization upon admission (aOR: 3.5; CI 1.2-10.1) and during ICU stay (aOR: 14.5; CI 2.1-100.1). Colonization by a multidrug pathogen could promote colonization by another.

Toxoplasma gondii meningoencephalitis without cerebral MRI findings in a patient with ulcerative colitis under immunosuppressive treatment.

Toxoplasmosis is the most common opportunistic infection of the central nervous system in immunosupressed patients. It is usually presented as a space-occupying lesion detected by cerebral computerized tomography or magnetic resonance imaging. The diffuse form of the disease (diffuse toxoplasmic meningoencephalitis) lacks the characteristic cerebral radiologic findings rendering pre-mortem diagnosis much more difficult. Herein, we describe a case of toxoplasmic menincoencephalitis, without evidence of cerebral space-occupying lesions, in a patient with ulcerative colitis under combined therapy with systemic glucocorticoids and azathioprine. Diagnosis was based on microscopic examination of cerebrospinal fluid (CSF) for the parasite, whereas, RT-PCR for Toxoplasma gondii was negative. Taking into consideration the limitations of molecular methods, investigation of the etiology of meningeal involvement in patients under immunosuppressive therapy presenting positive serology of previous T. gondii infection, should include microscopic examination of CSF for parasite presence.

Virulence factors among Staphylococcus lugdunensis are associated with infection sites and clonal spread.

Staphylococcus lugdunensis has emerged as a significant human pathogen, with distinct clinical and microbiological characteristics. Our goal was to identify the virulence factors in S. lugdunensis recovered from infected patients of two Greek hospitals during a six-year period (2008-2013). A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. Strains were classified into pulsotypes by pulsed-field gel electrophoresis (PFGE) of SmaI DNA digests. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from prosthetic device-associated infections (PDAIs). All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The highest resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, whereas 26/38 (68.4%) carried the ica operon. Biofilm formation was more frequent in isolates from PDAIs. Thirty-six strains (94.7%) carried atlL and 31 (81.6%) carried vwbl, whereas slush was detected in 15 (39.5%). PFGE revealed a low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones (C: 22 and D: nine strains). Type C strains recovered from all infection sites prevailed in biofilm formation and ermC carriage, whereas type D strains associated with SSTIs and DSIs carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, the clonal expansion and carriage of virulence factors, combined with biofilm-producing ability, render this species an important pathogen that should not be ignored.

Risk factors for enterococcal infection and colonization by vancomycin-resistant enterococci in critically ill patients.

PURPOSE: Vancomycin-Resistant Enterococci (VRE) are important causes of Intensive Care Unit (ICU) infections. Our goal was to identify the prevalence and risk factors for VRE colonization upon ICU admission and during ICU stay, as well as, their impact in enterococcal infection including vancomycin-susceptible cases (VSE). METHODS: A prospective study regarding patients admitted in ICU (n = 497) was conducted during a 24-month period. Rectal swabs were collected upon admission and during hospitalization and inoculated onto selective medium. Enterococci were phenotypically characterized. van genes were investigated by PCR and clones were identified by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. Epidemiologic data were collected from the ICU database. RESULTS: Risk factors for VRE carriage upon ICU admission (71/497) were: duration of previous hospitalization, glycopeptide administration, chronic heart failure, malignancy, insulin-dependent diabetes mellitus, and previous enterococcal infection (VRE and/or VSE). Risk factors for VRE colonization during ICU stay (36/250) were: quinolone administration, chronic obstructive pulmonary disease, chronic renal failure, and number of VRE-positive patients in nearby beds. Risk factors for enterococcal infection during ICU stay (15/284), including VRE and VSE cases, were: administration of third- or fourth-generation cephalosporins, cortisone use before ICU admission and VRE colonization, whereas, enteral nutrition was a protective factor. CONCLUSIONS: Previous VRE colonization and antibiotic usage are essential parameters for enterococcal infection (by VRE or VSE) during ICU stay. Previous enterococcal infection, co-morbidities and antibiotic usage are associated with VRE colonization upon ICU admission, whereas, patient to patient transmission, co-morbidities and antibiotic usage constitute risk factors for VRE colonization during ICU hospitalization.

The role of colonization pressure in the dissemination of colistin or tigecycline resistant KPC-producing Klebsiella pneumoniae in critically ill patients.

PURPOSE: To identify the risk factors for incident enteric colonization by KPC-producing Klebsiella pneumoniae (KPC-Kp) resistant to colistin or tigecycline during Intensive Care Unit (ICU) stay. METHOD: A prospective observational study of patients admitted to the ICU was conducted during a 27-month period. Rectal samples taken upon admission and weekly afterwards were inoculated on selective chromogenic agar. K. pneumoniae isolates were characterized by standard methodology. Mean inhibitory concentration (MIC) to colistin and tigecycline were determined by E-test. The presence of bla KPC gene was confirmed by PCR. RESULTS: Among 254 patients, 62 (24.4%) became colonized by colistin- resistant KPC-Kp during their stay. Multivariate analysis revealed that corticosteroid, colistin administration and number of colonized patients in nearby beds per day were significantly associated with colonization. Among 257 patients, 39 (17.9%) became colonized by tigecycline resistant KPC-Kp during their stay. Risk factors identified by multivariate analysis were: days at risk, obesity, number of colonized patients treated in nearby beds per day and administration of tigecycline. CONCLUSIONS: The high prevalence of colistin or tigecycline resistant KPC-Kp enteric carriage in ICU patients indicate that dissemination is due to their transfer from patient to patient via the personnel and indicates the importance of strict infection control protocols.

The combined effect of surface chemistry and flow conditions on Staphylococcus epidermidis adhesion and ica operon expression.

The assessment of biomaterial susceptibility to infection relies mainly on the analysis of macroscopic bacterial responses to material interactions, usually under static conditions. However, new technologies permit a more profound understanding of the molecular basis of bacteria-biomaterial interactions. In this study, we combine both conventional phenotypic analysis - using confocal microscopy - and genotypic analysis - using the relative reverse transcription polymerase chain reaction (RT-PCR) - to examine the interaction of bacteria with OH- and CH3-terminated glass surfaces, under dynamic flow conditions. Bacterial adhesion, as well as slime production and biofilm formation, was much higher on the CH3-terminated than on the OH-terminated glass - for four Staphylococcus epidermidis strains. This was in agreement with the icaA and icaD gene expression results that showed increased expression for the bacteria adhering to the CH3-terminated substrate, especially under the higher shear rate. Therefore, the combined effect of the surface chemistry and shear significantly influence the adhesion and phenotype of interacting bacterial cells, while there are putative links between phenotypic responses to bacteria-material interactions and gene-expression profile alterations. This indicates that analysis of gene expression not only can greatly refine our knowledge of bacteria-material interactions, but also yield novel biomarkers for potential use in biocompatibility assessment.

The first case of Staphylococcus aureus ST398 causing bacteremia in an immunocompromised patient in Greece.

We describe a case of catheter-related bloodstream infection, in a patient with colon cancer, caused by a methicillin-sensitive Staphylococcus aureus strain, nontypeable by pulsed field gel electrophoresis of SmaI macrorestriction fragment analysis, belonging to ST398. The patient recovered after daptomycin therapy. This is the first report that documents the emergence of ST398 in Greece.

Neonatal intensive care unit candidemia: epidemiology, risk factors, outcome, and critical review of published case series.

Evaluation of epidemiological trends, risk factors, and clinical outcome associated with candidemia at a neonatal intensive care unit is reported. From January 2005 to December 2009, forty candidemia cases were identified. C. albicans and C. parapsilosis were the most common species recovered (69 and 24%, respectively). All C. parapsilosis strains were susceptible to antifungals, whereas, C. albicans exhibited higher resistance rates to azoles. Low birth weight, low gestational age, presence of central lines, endotracheal intubation, total parenteral nutrition, previous use of antibiotics, steroids, previous episode(s) of bacteremia and prolonged stay in intensive care unit were common features associated with candidemia. C. albicans was most often isolated from extremely low birth weight neonates as compared to non-albicans Candida (P < 0.01). Mortality rate was 35.7% and was associated with low gestational age (P < 0.01), low birth weight (P < 0.01), and presence of renal failure (P < 0.05). Furthermore, a critical review of recent published case series is presented.

Changes of dermatophytoses in southwestern Greece: an 18-year survey.

The isolation and distribution rate of dermatophytes as causative agents of superficial mycoses of skin, hair, and nails during an 18-year period (1991-2008) at a university hospital are presented. A comparative analysis of epidemiological differences within the first (1991-1999) and the second 9-year period (2000-2008) was performed. Skin scrapings, nail, and hair specimens were examined by a direct microscopic examination and culture. Identification of dermatophyte species was based on macroscopic and microscopic characteristics of colonies. During the complete period (18 years), 5,971 patients with suspected dermatophytosis were examined. Seven hundred and sixty-nine patients (12.8%) were found positive. Among them, 495 cases (64.3%) were of skin dermatophytoses, 91(11.8%) of hair, and 183 (23.7%) of nails. The most frequent etiological agents were Microsporum canis (54%), Trichophyton rubrum (38%), and T. mentagrophytes (6%). Epidermophyton floccosum, T. tonsurans, T. violaceum, and M. gypseum were responsible only for 16 cases (2%) of dermatophytoses. The prevalence of dermatophytoses seems to decrease significantly from 16.2% (1991-1999)-9.6% during the last 9-year period. The most frequent dermatophyte, M. canis, shows decreasing trends during the last period (from 58.5 to 45.7%), whereas T. rubrum shows an increasing isolation rate (from 35 to 43.6%), respectively. The most common form of dermatophytosis among children remains tinea capitis due to M. canis. The most frequent etiological agent of tinea unguium (81%) is T. rubrum.

Ocular surface bacterial colonisation in sedated intensive care unit patients.

We investigated the time-dependent ocular surface bacterial colonisation of sedated patients hospitalised in an intensive care unit and aimed to evaluate whether proper topical antibiotic prophylaxis could prohibit corneal infection. The study lasted 12 months and included 134 patients undergoing sedation and mechanical respiratory support for various medical reasons. Patients hospitalised for less than seven days and those with pre-existing ocular surface pathology were excluded. All patients were examined on admission by inspecting the cornea for erosions. Followup examinations were performed each subsequent day. Cultures were also obtained from the conjunctival sac of both eyes on admission and every seventh day until the end of sedation. Standard laboratory techniques were used for isolation, identification and antibiotic susceptibility testing of bacteria. Antibiotic treatment for prophylaxis was administered accordingly. Analysis was carried out for 70 patients. Duration of sedation ranged from seven to 122 days. Fifty-four (77%) patients were colonised by at least one bacterial species other than normal flora within seven to 42 days. Multiple bacteria were isolated from 28 patients undergoing prolonged sedation. Prevalent isolates were Pseudomonas aeruginosa, Acinetobacter spp. and Staphylococcus epidermidis. Infectious keratitis was prohibited in all cases. Ocular surface of long-term sedated patients was found to be colonised by various bacterial species and their isolation was closely associated with the time period of hospitalisation. The results of this study suggest that the early identification of ocular surface bacteria colonisation and the administration of topical antibiotics for prophylaxis can prohibit corneal infection in these patients.