Similar bacterial signatures in the gut microbiota of type 1 and type 2 diabetes patients and its association with G protein-coupled receptor 41 and 43 gene expression.

Purpose: There are conflicting reports regarding the abundance of short-chain fatty acids producing bacteria in the gut microbiota in patients with type 1 and type 2 diabetes. We aimed to determine the amount of Akkermansia muciniphila, Anaerobutyricum hallii, Bifidobacterium adolescentis, Bifidobacterium longum, Collinsella aerofaciens, Faecalibacterium prausnitzii, Lacticaseibacillus rhamnosus, and Parabacteroides distasonis in the gut microbiota in patients with type1 and type2 diabetes, compared with the healthy controls and analyze the correlation between the gene expression levels of two short-chain fatty acids receptors GPR41 and GPR43. Methods: Forty type 1, 40 type 2 stool and blood samples of diabetes patients, and 40 healthy control samples were studied. DNA and RNA were extracted, and bacteria were detected using a Microbial DNA qPCR Assay kit. Gene expressions were detected with GPR41 and GPR43 primers via in-house qPCR. Results: Compared with healthy controls, B.longum and F.prausnitzii abundance were significantly decreased in patients with type1 and type2 diabetes, A.hallii abundance was increased in patients with type1 and decreased in type2 diabetes contrarily A.muciniphila abundance was decreased in patients with type1 and increased in type2 diabetes. GPR43 gene expression was upregulated in both patients group, however GPR41 was upregulated only in patients with type2 diabetes. Conclusions: Elevated B. longum and F. prausnitzii abundances were detected in the gut microbiota of patients with type1 and type2 diabetes and compared with healthy controls. B. longum and F.prausnitzii abundances were also correlated with the GPR43 gene expression level in type1 diabetes patients. Extensive studies determining bacteria producing short-chain fatty acids in gut microbiota, and their contribution in the pathogenesis of diabetes, are needed to understand better the mechanism of these diseases.

Firmicutes/Bacteroidetes Ratio in the Gut Microbiota and IL-1ß, IL-6, IL-8, TLR2, TLR4, TLR5 Gene Expressions in Type 2 Diabetes.

BACKGROUND: The aim of this study was to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota and IL-1ß, IL-6, IL-8, TLR2, TLR4 and TLR5 gene expression levels in the blood of adult type 2 diabetes (T2D) patients and compare it with that of adult nondiabetic healthy controls (HC). METHODS: Between May 2016 and April 2017, 99 T2D patients and 99 HCs were enrolled in the study. Bacteroidetes and Firmicutes levels were assessed from stool sample DNA and IL-1ß, IL-6, IL-8, TLR2, TLR4, and TLR5 gene expression levels assesed from blood sample RNA via qPCR from both T2D patients and healthy controls. RESULTS: The Firmicutes/Bacteroidetes ratio detected in the stool of type 2 diabetes patients was found to be higher with a statistically significant difference (p < 0.0001). Gene expression levels of IL-1ß, IL-6, IL-8, TLR2, TLR4, and TLR5 were found to be upregulated. CONCLUSIONS: The highest upregulation was detected in IL-6 with 11 fold in T2D patients comparing with HCs. F/B ratio and gene expression levels were elevated in T2D patients. Firmicutes were positively correlated with studied gene expressions. A better understanding of the complex interaction between gut microbiota, environment, and diabetes will allow for more effective prevention and treatment strategies for T2D.

A 5-year surveillance of healthcare-associated infections in a university hospital: A retrospective analysis.

Objectives: "Nosocomial infections" or "healthcare-associated infections" are a significant public health problem around the world. This study aimed to assess the rate of laboratory-confirmed healthcare-associated infections, frequency of nosocomial pathogens, and the antimicrobial resistance patterns of bacterial isolates in a University Hospital. Methods: A retrospective evaluation of healthcare-associated infections in a University Hospital, between the years 2015 and 2019 in Tekirdag, Turkey. Results: During the 5 years, the incidence densities of healthcare-associated infections in intensive care units and clinics were 10.31 and 1.70/1000 patient-days, respectively. The rates of ventilator-associated pneumonia, central line-associated bloodstream infections, and catheter-associated urinary tract infections in intensive care units were 11.57, 4.02, and 1.99 per 1000 device-days, respectively. The most common healthcare-associated infections according to the primary sites were bloodstream infections (55.3%) and pneumonia (20.4%). 67.5% of the isolated microorganisms as nosocomial agents were Gram-negative bacteria, 24.9% of Gram-positive bacteria, and 7.6% of Candida. The most frequently isolated causative agents were Escherichia coli (16.7%) and Pseudomonas aeruginosa (15.7%). The rate of extended-spectrum beta-lactamase production among E. coli isolates was 51.1%. Carbapenem resistance was 29.8% among isolates of P. aeruginosa, 95.1% among isolates of Acinetobacter baumannii, and 18.2% among isolates of Klebsiella pneumoniae. Colistin resistance was 2.4% among isolates of A. baumannii. Vancomycin resistance was 5.3% among isolates of Enterococci. Conclusion: Our study results demonstrate that healthcare-associated infections are predominantly originated by intensive care units. The microorganisms isolated from intensive care units are highly resistant to many antimicrobial agents. The rising incidence of multidrug-resistant microorganisms indicates that more interventions are urgently needed to reduce healthcare-associated infections in our intensive care units.

The incidence of acute kidney injury and its association with mortality in patients diagnosed with COVID-19 followed up in intensive care unit.

INTRODUCTION: The kidneys are some of the most frequently affected organs during coronavirus disease 2019 (COVID-19). This multicenter study evaluated the incidence of and risk factors for acute kidney injury (AKI) in COVID-19 patients followed up in intensive care unit (ICU) and its association with mortality. METHODS: Three hundred twenty-eight patients diagnosed with COVID-19 and hospitalized in ICU were included. Risk factors associated with AKI and mortality were evaluated. RESULTS: Eighty-eight patients (27.9%) were diagnosed with AKI. AKI was significantly associated with older age, higher baseline creatinine level, lower albumin level, and coexistence of cardiovascular disease and chronic obstructive pulmonary disease. Mortality in the entire study group was significantly associated with AKI, older age, requirement of invasive mechanical ventilation, higher neutrophil level, lower lymphocyte, and albumin levels. CONCLUSION: AKI is frequently seen during the course of COVID-19 and is associated with high mortality. Identifying AKI-related risk factors appears essential in the management of COVID-19 patients.

Burkholderia cepacia complex bacteremia outbreaks among non-cystic fibrosis patients in the pediatric unit of a university hospital.

BACKGROUND: Burkholderia cepacia complex (Bcc) comprises multi-drug resistant, Gram-negative, motile, and aerobic bacteria. Bcc causes severe nosocomial infections particularly in patients with intravascular catheters and in those with cystic fibrosis. We studied a Bcc outbreak in non-cystic fibrosis patients. METHODS: We analyzed data from six patients hospitalized at our center. Blood cultures identified as infectious were incubated onto 5% blood sheep agar, chocolate agar, and eosin methylene blue (EMB) agar. We examined possible sites that could be sources of infection at the clinic. We confirmed isolations with pulsed-field gel electrophoresis (PFGE) tests. RESULTS: The first patient was hospitalized due to left renal agenesis, urinary tract infection, and renal failure. Bcc was isolated in blood cultures obtained due to high fever on the third day of hospitalization. We stopped new patient hospitalizations after detecting Bcc in blood cultures of other five patients. We did not detect further positive specimens obtained from other clinic and the patient rooms. PFGE patterns were similar in all clinical isolates of Bcc indicating that the outbreak had originated from the source. CONCLUSIONS: Bcc infection should be considered in cases of nosocomial outbreaks of multi-drug resistant organisms that require hospitalization at intensive care units. Control measures should be taken for prevention of nosocomial infections and required investigations should be done to detect the source of infection.

Effects of calcineurin inhibitors, cyclosporine A and tacrolimus (FK506), on the activity of antifungal drugs against Candida spp.

Introduction. The simultaneous use of antifungals with immunosuppressive agents has become a necessity for patients taking immunosuppressive therapy. However, antifungal drugs are problematic because of their limited target.Hypothesis. Scientists have been searching for new antifungals and some compounds with at least additive effects on antifungals. Calcineurin inhibitors used as immunosuppressive agents also attract attention due to their antifungal property.Aim. To evaluate the activity of two calcineurin inhibitors alone and in combination with amphotericin B (AMB), caspofungin (CAS), itraconazole (ITR), voriconazole (VOR) and fluconazole (FLU).Methodology. MICs of AMB, CAS, ITR, VOR, FLU and cyclosporine A (CsA) and tacrolimus (TAC) as calcineurin inhibitors were evaluated by the broth microdilution method against Candida albicans (n=13), C. krusei (n=7) and C. glabrata (n=10). Checkerboard and time-kill methods were performed to investigate the activity of combining calcineurin inhibitors with antifungal drugs.Results. The lowest MIC values were detected with VOR for all Candida isolates tested. Although we did not detect any inhibition for CsA or TAC alone at concentrations tested in this study, the combinations of CAS with CsA showed the highest synergistic activity (36.7%) by the checkerboard method, and CAS with CsA and ITR with TAC combinations exhibited apparent synergistic interaction by the time-kill method. However, the combinations of both CsA and TAC with AMB resulted in antagonistic interactions, especially against C. krusei isolate in time-kill testing.Conclusion. Synergistic interactions in the combinations of TAC or CsA with antifungal drugs, except for AMB, in many concentrations was found to be promising in terms of the treatment of patients with fungal infections.

Comparison of the Sensititre YeastOne antifungal method with the CLSI M27-A3 reference method to determine the activity of antifungal agents against clinical isolates of Candidaspp.

Background and aim: Infections caused by Candida species are significantly increasing today, and invasive Candida infections are generally associated with high mortality. Early diagnosis and identification of Candida spp. is important for the determination of antifungal agents that will be used for treatment. The aim of the present study was to provide a better regimen for Candida infections in the future. Materials and methods: TheSensititre YeastOne (SYO) method was compared with The Clinical Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) testing method. Endpoints of minimal inhibitory concentrations (MICs) were determined for both methods. Results: By using both methods, MIC values of micafungin, caspofungin, voriconazole, and fluconazole were lower than amphotericin B. The values obtained with the SYO method were in high categorical agreement for ecinocandins and amphotericin B. The results of voriconazole and fluconazole were in low categorical agreement. The categorical agreement between the SYO and the BMD results at 24 h was 82.1% for VORI and 98.4% for AMB. Values obtained with SYO method for all antifungal agents were in high essential agreement with the data of the CLSI reference BMD method. The essential agreement between the SYO and the BMD results at 24 h was 94.0% for MFG and 99.0% for AMB. Conclusions: The SYO method was ready-to use, so it appeared to be easier and more efficient for Candida isolates.

Bacteroidetes and Firmicutes levels in gut microbiota and effects of hosts TLR2/TLR4 gene expression levels in adult type 1 diabetes patients in Istanbul, Turkey.

AIM: The aim of this study was to determine and compare the levels of both Bacteroidetes and Firmicutes in the gut microbiota and TLR2/TLR4 gene expression in the blood of patients with type 1 diabetes mellitus (T1DM) and healthy individuals. These results may serve as a preliminary assessment to guide future research. METHOD: Between January and October 2014, stool and blood samples were collected from 53 adult T1DM patients and 53 age- and gender-matched healthy individuals. Bacteroidetes and Firmicutes levels were assessed from stool sample DNA and TLR2 and TLR4 expression levels were analyzed via qPCR using RNA from EDTA blood samples from both patients and healthy controls. RESULTS: The amounts of Bacteroidetes and Firmicutes were statistically significantly higher and lower, respectively, in the T1DM group than in the healthy control group (p < 0.001 and p < 0.001, respectively). In addition, the Firmicutes/Bacteroidetes ratios in patients with T1DM were significantly lower than in healthy controls. The TLR4 and TLR2 gene expression levels in T1DM patients were significantly upregulated and downregulated, respectively, compared to those in the control group. CONCLUSION: Our data are the first to show a relationship between T1DM and gut microbiota in our country. In addition, our results provide information about the connections between T1DM, gut microbiota, and TLR2 and TLR4 expression. We believe that Bacteroidetes and Firmicutes in the gut microbiota may play a role in the autoimmune process of T1DM and that these findings should be further investigated in the future.

An updated comprehensive systematic review of Cladophialophora bantiana and analysis of epidemiology, clinical characteristics, and outcome of cerebral cases.

Cladophialophora bantiana is a phaeoid fungus that only rarely has been isolated from sources other than the human brain. It has a particular tropism for the central nervous system (CNS). We have integrated and updated large-scale data related to several aspects of C. Bantiana and reviewed all the available reports on its cerebral infections, focusing on their geographical distribution, infection routes, immune status of infected individuals, type and location of infections, clinical manifestations and treatment and outcome, briefly looking over the spectrum of other disease entities associated with C. bantiana, that is, extra-cerebral and animal infections and on the environmental sources of this fungus. Among the agents of phaeohyphomycosis, a term used to describe an infection caused by a dark pigmented fungus, C. bantiana has some significant specific features. A total of 120 case reports were identified with a significantly higher percentage of healthy subjects than immune-debilitated patients (58.3% vs. 41.7%). Infections due to C. bantiana occur worldwide. The main clinical manifestations are brain abscess (97.5%), coinfection of brain tissue and meninges (14.2%) and meningitis alone (2.5%). Among immunocompetent patients, cerebral infection occurred in the absence of pulmonary lesions. The mortality rate is 65.0% regardless of the patient's immune status. The therapeutic options used include surgery or antifungals alone, and the combination of both, in most cases the fatal outcome being rapid after admission. Since the fungus is a true pathogen, laboratory workers should be made aware that BioSafety Level-3 precautions might be necessary.

A case of central nervous system infection due to Cladophialophora bantiana.

BACKGROUND: Cladophialophora bantiana is a melanised mold with a pronounced tropism for the central nervous system, almost exclusively causing human brain abscesses. CASE REPORT: We describe a case of cerebral infection by this fungus in an otherwise healthy 28-year-old coal-miner. Environmental occurrence, route of entry, and incubation period of this fungus are unknown, but our case is informative in that the first symptoms occurred about eight weeks after known traumatic inoculation. Lesions were compatible with tuberculous granulomas, and the patient initially received antitubercular treatment. Melanised fungal cells were seen in a brain biopsy and abscess materials. Therapy was switched from empirical antitubercular treatment to amphotericin B (0.5mg/kg/d), but was changed to voriconazole 200mg/d, i.v. on the basis of antifungal susceptibility test results. The patient responded clinically, and gradually improved. The isolate was identified by sequencing of the Internal Transcribed Spacer domain of rDNA. CONCLUSIONS: Given the non-specific clinical manifestations of C. bantiana cerebral abscesses, clinicians and laboratory workers should suspect infections caused by C. bantiana, particularly in immunocompromised patients with a trauma history.

Evaluation of Virulence Factors and Antifungal Susceptibility in Yeast Isolates from Postmortem Specimens.

Invasive fungal infections are a leading cause of morbidity and mortality in immunocompromised patients, especially in cases requiring a prolonged stay in the intensive care unit. A total of 99 yeast strains were isolated from 42 postmortem cases. In this study, virulence factors and antifungal susceptibility of these species were evaluated. The isolates were identified as Candida albicans (54), C. tropicalis (15), C. glabrata (12), C. parapsilosis (6), C. lipolytica (3), C. utilis (3), C. krusei (2), C. kefyr (1), and Cryptococcus neoformans (3). The most commonly isolated species was C. albicans, and no resistant species were determined. Despite the equal number of specimens, no secretion of significant virulence factors was associated with the postmortem specimen in the Candida species. Postmortem fungal investigations in forensic autopsies are useful in explaining cause of death in such cases, also may lead to protocols for the treatment of fungal infections and contribute to fungal pathogenesis and epidemiological data.

Virulence Attributes and Antifungal Susceptibility Profile of Opportunistic Fungi Isolated from Ophthalmic Infections.

Investigations of both virulence factors and antifungal susceptibility profiles are crucial for understanding the pathogenesis and prognosis of ophthalmic mycoses. In this study, we investigated the in vitro antifungal susceptibility of amphotericin B (AMB), voriconazole (VRC), and natamycin (NAT) against a set of 50 fungal isolates obtained from patients with ocular mycoses using the Clinical and Laboratory Standards Institute broth microdilution method. In addition, putative virulence factor, such as secretory phospholipases and proteinases, and biofilm formation activity were analyzed. The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of the antifungals across all isolates were the following (in increasing order): VRC (0.70 µg/mL), AMB (0.81 µg/mL), and NAT (1.05 µg/mL). The highest activity against 14 Aspergillus strains was exhibited by VRC (GM MIC: 0.10 µg/mL), followed by AMB and NAT (GM MICs: 0.21 and 0.27 µg/mL), respectively. However, for 12 Fusarium spp., the GM MIC of VRC (2.66) was higher than those of NAT and AMB (GM MICs 1.3 and 0.8 µg/mL, respectively). Proteinase and phospholipase activity were observed in 30 % and 42 % of the isolates, respectively, whereas only 8 % of the isolates were able to produce biofilms. Phospholipase activity was observed in all Fusarium isolates, but not in any of the Aspergillus isolates. In contrast, biofilm-forming capability was detected in 25 % of the Fusarium isolates, but none of the Aspergillus isolates. The differences in the MICs of AMB, VRC, and NAT, biofilm-forming ability and proteinase and phospholipase activities among the isolates were not significant (p > 0.05). Overall, our study suggests no significant correlation between the antifungal susceptibility profiles and virulence attributes of ocular fungal isolates.

Virulence markers of opportunistic black yeast in Exophiala.

The black yeast genus Exophiala is known to cause a wide variety of diseases in severely ill individuals but can also affect immunocompetent individuals. Virulence markers and other physiological parameters were tested in eight clinical and 218 environmental strains, with a specific focus on human-dominated habitats for the latter. Urease and catalase were consistently present in all samples; four strains expressed proteinase and three strains expressed DNase, whereas none of the strains showed phospholipase, haemolysis, or co-haemolysis activities. Biofilm formation was identified in 30 (13.8%) of the environmental isolates, particularly in strains from dishwashers, and was noted in only two (25%) of the clinical strains. These results indicate that virulence factors are inconsistently present in the investigated Exophiala species, suggesting opportunism rather than pathogenicity.

Microbiota-Gut-Brain Axis: Yeast Species Isolated from Stool Samples of Children with Suspected or Diagnosed Autism Spectrum Disorders and In Vitro Susceptibility Against Nystatin and Fluconazole.

Autism spectrum disorder (ASD) is a general term for a group of complex neurodevelopmental disorders of brain development that limits a person's ability to function normally. Etiology has not been clearly defined up to date. However, gut microbiota and the bidirectional communication between the gastrointestinal tract and brain, the so-called microbiota-gut-brain axis, are hypothesized, which may be involved in the etiology of several mental disorders. Recent reports suggest that Candida, particularly Candida albicans, growth in intestines may cause lower absorption of carbohydrates and minerals and higher toxin levels which are thought to contribute autistic behaviors. The aim of this study was to identify the 3-year deposited yeasts isolated from stool samples of children with diagnosed or suspected ASD and to determine in vitro activity of nystatin and fluconazole against these isolates using Clinical Laboratory Standards Institute M27-A3 guidelines. A 17-year retrospective assessment was also done using our laboratory records. Among the species identified, intrinsically fluconazole-resistant Candida krusei (19.8 %) and Candida glabrata (14.8 %) with elevated MICs were remarkable. Overall, C. albicans (57.4 %) was the most commonly isolated species in 17 years. The species identification and/or antifungal susceptibility tests have to be performed using the strain isolated from stool sample, to select the appropriate antifungal agent, if antimycotic therapy is needed.

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses.

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (E max) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5-1.0, 1.0, 0.5-1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0-4.0, 0.5, 1.0 for A. flavus, and 0.12-0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0-16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03-0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (E(max) = 0.63 h(-1), r(2) = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (E(max) = 0.39 h(-1), r(2) = 0.87), voriconazole (E(max) = 0.35 h(-1), r(2) = 0.098), and itraconazole (E(max) = 0.01 h(-1), r(2) = 0.98). Overall, the antifungal susceptibility of the non-fumigatus Aspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.

Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi?

Although conventional identification of pathogenic fungi is based on the combination of tests evaluating their morphological and biochemical characteristics, they can fail to identify the less common species or the differentiation of closely related species. In addition these tests are time consuming, labour-intensive and require experienced personnel. We evaluated the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeast and 160 moulds in our clinical mycology laboratory. While the yeast isolates were identified at species level with 100% homology, 102 (63.75%) clinically important mould isolates were identified at species level, 56 (35%) isolates at genus level against fungal sequences existing in DNA databases and two (1.25%) isolates could not be identified. Consequently, Whatman FTA filter matrix technology was a useful method for extraction of fungal DNA; extremely rapid, practical and successful. Sequence analysis strategy of D1-D2 region of the large ribosomal subunit gene was found considerably sufficient in identification to genus level for the most clinical fungi. However, the identification to species level and especially discrimination of closely related species may require additional analysis.

The role of Porphyromonas gingivalis in the development of atherosclerosis and its relationship with fim A genotype.

BACKGROUND: Porphyromonas gingivalis, a major periodontal pathogen, is gaining increasing attention for its possible association with atherosclerosis. Its fimbriae are classified into six genotypes (Types I-V, Ib) based on the diversity of the fim A genes encoding the fimbrial subunits. In this study, fim A genotype's distribution of P. gingivalis was analyzed in atherosclerotic plaque specimens. METHODS: A total of 50 atherosclerotic plaque specimens and 50 non-atherosclerotic, post stenotic aneurysm specimens were collected from patients undergoing cardiovascular surgery. Bacterial DNA was also extracted from each specimen, as real-time PCR was carried out with P. gingivalis-specific primer sets. The positive specimens of P. gingivalis were further analyzed to discriminate the fim A genotype using real-time and nested PCR methods. RESULTS: P. gingivalis was detected only in one atherosclerotic plaque; however, the genotype was nontypable in this specimen. CONCLUSIONS: We state that it is not easy to show a significant relationship between P. gingivalis, its fim A genotype, and atherosclerosis. We suggest that new extended studies based especially upon the quantitave determination of P. gingivalis and its genotype distribution on atherosclerotic specimens are needed to show an evident relationship between atherosclerosis and P. gingivalis.

First imported coccidioidomycosis in Turkey: A potential health risk for laboratory workers outside endemic areas.

Coccidioidomycosis caused by Coccidioides immitis or Coccidioides posadasii is endemic in arid climate zones in America, travel-related cases have been reported. We report the first documented case of coccidioidomycosis in Turkey, overviewing reported cases in Europe and underlying difficulties of differential diagnosis outside endemic regions. The patient was an otherwise healthy 41-year-old man who travelled endemic areas. Laboratory diagnosis was based on direct microscopy of two subsequent subcutaneous biopsy specimens and culture and confirmed molecularly. Laboratory personnel should become aware that BioSafety Level-3 organisms may become more frequent and widespread.

The usefulness of DNA sequencing after extraction by Whatman FTA filter matrix technology and phenotypic tests for differentiation of Candida albicans and Candida dubliniensis.

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.

Prevalence and antimicrobial susceptibilities of anaerobic bacteria isolated from perforated corneal ulcers by culture and multiplex PCR: an evaluation in cases with keratitis and endophthalmitis.

BACKGROUND: Anaerobic bacteria play an important role in eye infections; however, there is limited epidemiologic data based on the the role of these bacteria in the etiology of keratitis and endophthalmitis. The aim of this re- search is to determine the prevalence of anaerobic bacteria in perforated corneal ulcers of patients with keratitis and endophthalmitis and to evaluate their antimicrobial susceptibilities. METHODS: Corneal scrapings were taken by the ophthalmologist using sterile needles. For the isolation of anaerobic bacteria, samples were inoculated on specific media and were incubated under anaerobic conditions obtained with Anaero-Gen (Oxoid & Mitsubishi Gas Company) in anaerobic jars (Oxoid USA, Inc. Columbia, MD, USA). The molecular identification of anaerobic bacteria was performed by multiplex PCR and the susceptibilities of an- aerobic bacteria to penicillin, chloramphenicol, and clindamycin were determined with the E test (bioMerieux). RESULTS: 51 strains of anaerobic bacteria belonging to four different genuses were detected by multiplex PCR and only 46 strains were isolated by culture. All of them were found susceptible to chloramphenicol whereas penicillin resistance was found in 13.3% of P.anaerobius strains, clindamycin resistance was found in 34.8% of P.acnes and 13.3% of P. anaerobius strains. Additionnaly, one strain of P. granulosum was found resistant to clindamycin, one strain of B. fragilis and one strain of P.melaninogenica were found resistant to penicillin and clindamycin. CONCLUSIONS: Routine analyses of anaerobes in perforated corneal ulcers is inevitable and usage of appropriate molecular methods, for the detection of bacteria responsible from severe infections which might not be deter- mined by cultivation, may serve for the early decision of the appropriate treatment. Taking into account the in- creasing antimicrobial resistance of anaerobic bacteria, alternative eye specific antibiotics effective against anaer- obes are needed to achieve a successful treatment.