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There is an urgent need to be able to identify carbapenemase-producing Enterobacterales. In this study we aimed to compare the performance of the MALDI Biotyper Selective Testing of Antibiotic Resi...

Background Antimicrobial resistance (AMR) is a growing problem worldwide, with an estimated high burden in low- and middle-income countries (LMICs). In these settings, tackling the problem of AMR is often constrained by a lack of reliable surveillance data due to limited use of microbiological diagnostics in clinical practice. Objectives The aim of this article is to present an overview of essential elements for setting up an AMR surveillance system in LMICs, to summarize the steps taken to develop such a system in the country of Georgia, and to describe its impact on microbiology laboratories. Sources A literature review of published papers using PubMed and experiences of experts involved in setting up AMR surveillance in Georgia. Content Basic requirements for implementing a laboratory-based surveillance system in LMICs can be captured under four pillars: (a) governmental support, (b) laboratory capacity and quality management, (c) materials and supplies, and (d) sample collection, data management, analysis and reporting. In Georgia, the World Health Organization Proof-of-Principle project helped to start the collection of AMR surveillance data on a small scale by promoting the use of microbiological diagnostics in clinics, and by providing training and materials for laboratories. Thanks to governmental support and a strong lead by the national reference laboratory, the AMR surveillance network was sustained and expanded after the project ended. Implications This review describes the Georgian approach in building and expanding a functional AMR surveillance system, considering the elements identified from the literature. The introduction of quality management systems, standardization of guidelines and training paired with targeted capacity building led to improved laboratory standards and management of patients with bloodstream infections. Reliable AMR surveillance data may inform and facilitate policy-making on AMR control. The Georgian experience can guide other countries in the process of building up their national AMR surveillance system.

Objectives: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardised antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. Methods: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. Results: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L–mm) were determined for amoxicillin-clavulanic acid (8–22), ceftazidime (8–22), imipenem (2–29), meropenem (2–26), doxycycline (2–none), tetracycline (8–23), chloramphenicol (8–22) and trimethoprim-sulfamethoxazole (4–28). Conclusions: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs, respectively, for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

There is an urgent need to be able to identify carbapenemase-producing Enterobacterales. In this study we aimed to compare the performance of the MALDI Biotyper Selective Testing of Antibiotic Resistance-βLactamase (MBT STAR-BL) test with the in-house Carba NP test in their ability to rapidly detect carbapenemase production in

Serious mycological work requires a reliable source of cultures that are maintained under safe long-term storage. In this study, 1186 clinical fungal isolates consisting of molds (20 species in 11 genera) and yeasts (21 species in seven genera) maintained in water, under mineral oil at room temperature and cryopreserved at -80 °C for periods ranging from 1 to 12 years, were evaluated for their viabilities and stabilities. The strains were subcultured onto either Sabouraud dextrose agar or potato dextrose agar to determine the viabilities and purities. The stabilities of the dermatophytes were investigated using urease test medium, the Trichophyton agar test and morphological examination. The stabilities of yeasts were evaluated by microscopic morphology and by determining the antifungal susceptibilities of random samples of yeasts (n = 120). Additionally, 365 strains (dermatophytes, n = 115; yeasts, n = 250) were further characterized by "matrix-assisted laser desorption/ionization time-of-flight mass spectrometry." After 12 years of preservation, the survival rates with the three different preservation techniques, i.e., in water, under mineral oil and by freezing, were assessed as 94.7, 82.0 and 97.4 %, respectively. Viability was generally unrelated to the duration of storage. More stable and consistent growth was achieved after storage in water and freezing compared with mineral oil preservation. Our results demonstrate that the procedure for maintaining fungal cultures in water is a simple and inexpensive method, next to cryopreservation, and that both can be reliably used for the long-term preservation of most fungal isolates.

In this study, the performance of the “RESIST-3 O.K.N. K-SeT” (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria ( n = 22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla KPC , bla IMP , bla VIM , bla NDM , and bla OXA-48 . The rates of bla NDM , bla OXA-48 , and bla KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla NDM and bla OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132 K. pneumoniae . The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli , Serratia marcescens , Citrobacter freundii , Enterobacter cloacae , K. pneumoniae carrying bla KPC , bla NDM , and/or bla OXA-48 . On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla IMP and bla VIM . We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.

Objectives To develop a rapid and simple method that can identify the presence of β-lactamases in clinical isolates and samples, and determine their activity on different types of β-lactam antibiotics, including carbapenems, within one hour. Methods In this study, we describe a thin layer chromatography-based method for rapid detection of β-lactamases including carbapenemases. The method relies on the examination of changes in the migration rate of β-lactams in chromatography, due to degradation by β-lactamase enzymes. A total of 44 isolates, 29 carbapenemase-producers and 15 non-carbapenemase-producers, were screened by this method. Results The method has proven to be able to distinguish β-lactamases as carbapenemase or non-carbapenemase producing strains with high sensitivity in one hour. Conclusions The method developed, provides information about the production of β-lactamases by bacteria and β-lactam drugs inactivated by these enzymes, including carbapenems. This new method may play an important role in guiding antimicrobial treatment, especially in critically ill patients infected bacteria producing β-lactamases.

Accurate and rapid identification of yeast isolates have become important in recent years for not only antifungal susceptibility testing due to the species-specific clinical resistance breakpoints but also early initiation of appropriate antifungal therapy. In clinical microbiology laboratories species identification of yeasts is often performed with several commercial systems based on biochemical properties and rarely according to the physiological and morphological characteristics. The aim of this study was to compare the two common commercial systems, VITEK 2 YST ID Card (Vitek; bioMérieux, France) and API 20C AUX (API; bioMérieux, France) with conventional mycological methods. A total of 473 clinical yeast strains isolated from clinical specimens in different university and training/research hospitals and identified by Vitek system were included in the study. The isolates were re-identified with API and conventional methods including morphological identification in the Mycology Reference Laboratory of the Public Health Institute of Turkey. Candida dubliniensis MYA 583, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32268 were used as quality control strains and those standard strains were studied consecutively 10 days with both of the methods. The results of identification by Vitek and API were compared with the results of conventional methods for those 473 yeast isolates [6 genus (Candida, Cryptococcus, Blastoshizomyces, Rhodotorula, Saccharomyces, Trichosporon), 17 species (5 common and 12 rarely isolated)]. The performances of the systems were better (Vitek: 95%; API: 96%) for the commonly detected species (C.albicans, C.parapsilosis, C.glabrata, C.tropicalis and C.krusei) than those for rarely detected species (Vitek: 78.4%; API: 71.6%) (p= 0.155). Misidentification or unidentification were mostly detected for C.parapsilosis (Vitek: 6/87; API: 7/87) and C.glabrata (Vitek: 9/104; API: 3/104) by both of the systems. For rarely detected yeast isolates, misidentification or unidentification were most frequently observed in species of C.pelliculosa (Vitek: 3/11; API: 6/11) and C.dubliniensis (API and Vitek: 2/5) isolates. Candida guilliermondii (API: 2/5) isolates had lower rate of identification with API compared to other species. Blastoschizomyces capitatus and Saccharomyces cerevisiae isolates could not be identified by both of the systems. As a result, the accurate diagnosis of Vitek and API systems were similar in terms of consistency (86.3%). Two systems performed well in correct identification of common clinical yeast species (at least 95%), while the identification of rare species was more challenging indicating that they require further morphological and physiological testing. The addition of morphological identification to commercial systems will be useful for accurate diagnosis and treatment of mixed infections.

We evaluated the usefulness of the Carba NP test for rapid detection of carbapenemase activity in Bacteroides spp. The minimum inhibitory concentration (MIC) for imipenem was determined with gradient test strips, and cfiA gene was investigated by polymerase chain reaction for 27 clinical Bacteroides spp. isolates. Carba NP test was performed according to recommendations of the Clinical and Laboratory Standards Institute. Among three cfiA gene harboring clinical isolates, two imipenem resistant isolates were Carba NP test positive, while the imipenem intermediate isolate was negative. Our preliminary results suggest that the Carba NP test can be useful as a rapid test to detect carbapenemases in Bacteroides species.

Microbiology laboratories are one of the major components of antimicrobial stewardship (AMS) programs. The early identification of pathogen and antimicrobial susceptibility tests helps physicians to direct the target specific therapy. Communication between laboratory staff and clinicians is essential to achieve the goals of antimicrobial stewardship program. This chapter summarizes the role of microbiology laboratory for successful AMS.

Hepatitis C infection is a public health problem. The aim of this retrospective study was to determine the distribution of hepatitis C virus (HCV) genotypes in seven regions of Turkey, by evaluating 7002 patients with chronic HCV in a six-year period. During the 2009–2014 period, serum/plasma samples from 7002 new consecutive HCV RNA positive patients were collected. The female patients were 3867 (55.2%). The genotype distribution of HCV patiens was evaluated by ages and years. Statistical analysis was performed by using the Mann–Whitney test and the ?2 analysis. During the six-year period, genotype 1b was the most common genotype (67.7%) followed by untypeable genotype 1 (7.7%), genotype 4 (7.3%) and genotype 3 (6.7%). In 2014, genotype 3 was the second most common one (11.3%) and genotype 4 was the third most common one (9.8%). In the group with

Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories.

Tüm dünyada Enterobacteriaceae ailesi üyeleri arasında çoklu ilaç direncine sahip izolatlar artış göstermekte ve tedavide son seçenek olarak kabul edilen karbapenemlere karşı bu izolatlarda bulunabilen karbapenemazlar tedavide başarısızlıklara yol açabilmektedir. Bu enzimlerin ülkemizdeki sıklıklarına ilişkin çok merkezli çalışmalar bulunmamaktadır. Bu çalışmanın amacı, European Survey on Carbapenemase Producing Enterobacteriaceae (EuSCAPE) projesi kapsamında, Türkiye'nin değişik bölgelerindeki merkezlerden izole edilen Escherichia coli ve Klebsiella pneumoniae izolatlarında karbapenemaz varlığını ve enzim tiplerini belirlemektir. Türkiye'de belirlenmiş 18 merkezden Kasım 2013 tarihinden itibaren ilk altı aylık süre içinde karbapenem grubu antibiyotiklerden (imipenem, meropenem veya ertapenem) en az birine dirençli olarak saptanan ilk 10 klinik izolat koordinatör merkeze gönderilmiştir. İzolatlarda imipenem, meropenem ve ertapenem antibiyotik duyarlılıkları mikrodilüsyon yöntemi ile EUCAST standartlarına göre araştırılmış ve karbapenemaz varlığı fenotipik olarak kombinasyon sinerji testi ile doğrulanmıştır. Karbapenemaz tipleri, VIM, IMP, NDM, KPC ve OXA-48 karbapenemaz genlerine özgül primerler kullanılarak multipleks-polimeraz zincir reaksiyonu yöntemi ile araştırılmıştır. Altı aylık süre içinde, koordinatör merkeze, karbapenemaz varlığı şüpheli 155 klinik izolat [21'i (%13.5) E.coli, 134'ü (%86.5) K.pneumoniae] gönderilmiştir. E.coli izolatlarının 19'unda (%90.5) ve K.pneumoniae izolatlarının 124'ünde (%92.5) olmak üzere toplam 155 izolatın 143'ünde (%92.3) genotipik olarak en az bir karbapenemaz geni saptanmıştır. Bu enzimler 136 izolatta tek başına (OXA-48: %84.6; NDM: %6.3; VIM: %2.8; IMP: %1.4) bulunurken, yedi izolatta iki enzim bir arada (OXA-48 + NDM: %2.1; OXA-48 + VIM: %2.1; VIM + NDM: %0.7) olarak tespit edilmiştir. İzolatların hiç birinde KPC enzimi gözlenmemiştir. Mikrodilüsyon testi sonuçlarına göre OXA-48 pozitif izolatların imipenem, meropenem ve ertapeneme direnç yüzdeleri sırasıyla %59.5, %52.9 ve %100 olarak belirlenmiştir. Karbapenemaz varlığının doğrulanması için yapılan kombinasyon sinerji testinin moleküler test ile %100 uyumlu olduğu saptanmıştır. OXA-48 enzimi içeren izolatların birçoğunun meropeneme duyarlı ancak tamamının ertapeneme dirençli olması nedeniyle, karbapenemaz taramasında ertapenemin en uygun antibiyotik olduğu, ekipman ve maliyet gerektiren moleküler yöntemlerin uygulanamadığı merkezlerde sinerji tekniğine dayalı bu fenotipik yöntemin güvenle kullanılabileceği sonucuna varılmıştır, Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confi rmed by combination disk synergy tests. Types of carbapenemases were investigated using specifi c primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available

Background: Recently, new carbapenemases in Enterobacteriaceae strains and non-fermentative gram-negative bacilli have been reported. The New Delhi metallo-beta-lactamase-1 (NDM-1) is a major problem around the world. The purpose of this article is to address the NDM-1 Klebsiella pneumoniae epidemic detected in eight cases in our hospital. Methods: Bacteria identified in this epidemic were from patients already admitted to the intensive care unit of the Sakarya University Training and Research Hospital during efforts toward establishment of infection surveillance and control program. Antimicrobial susceptibility testing of strains was performed using the VITEK 2 system (bioMerieux, France), E-test gradient strips (bioMerieux, France), and the disc diffusion test. For the metallo-beta-lactamase activity, the combined disc diffusion test and modified Hodge test as phenotypic tests were performed. To identify the resistance gene, the Xpert Carba-R kit (Cepheid Inc., USA) and an in-house multiplex polymerase chain reaction (PCR) method designed for five common carbapenemase genes (IMP, VIM, KPC, NDM-1, and OXA-48) were employed. The clonal relationship of these strains was explored by the repetitive PCR (rep-PCR, DiversiLab System, bioMerieux, France) method. Results: During the December 2014 to March 2015 period, NDM-1 positive K. pneumoniae strains were detected in eight patients. All of these strains were found to produce NDM-1, while two of them also revealed the presence of OXA-48. The rep-PCR results reveal a clonal proximity of 95 \% for six of the eight strains. Conclusions: Our findings suggest the tendency of NDM-1-producing strains to spread in our country as well. A carbapenem-resistant K. pneumoniae threat may pose a great risk to our country. It is clear that more comprehensive infection control precautions should be implemented in our hospitals.

WOS: 000371499500003, PubMed ID: 27058326, Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confirmed by combination disk synergy tests. Types of carbapenemases were investigated using specific primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available.

In one-third of the patients with amoebiasis, amoebic liver abscess (ALA) may occur after the penetration of amoebic trophozoites through the intestinal wall. ALA is seen mostly among men aged 20-45 years with a serious clinical outcome, with fever and abdominal pain on the right upper quadrant. Most patients have no recent history of amoebic colitis; indeed, they have neither gastrointestinal complaints nor Entamoeba histolytica (E. histolytica) cysts/trophozoites in their stools. Therefore, ultrasonography and serology are primary in ALA diagnosis, while searching for E. histolytica DNA in abscess fluid using PCR has been preferred as an effective and reliable method, lately. Early antimicrobial therapy is effective; however, for cases irresponsive to therapy after 72 hours and with large abscess, drainage or surgical intervention is indicated. If left untreated, ALA may disseminate to other organs and cause death. The data concerning the extra-intestinal manifestations of amebiasis in Turkey are limited. Here, a rare case of a young man with an initial diagnosis of pneumonia followed by the identification of ALA after radiological interventions and laboratory tests is presented and the relevant literature is discussed.

Background: Matrix–assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is known as a successful tool for bacterial identification and suitable for clinical diagnostics. The aim of our study was to evaluate diagnostic accuracy of MALDI-TOF MS system and VITEK2 Gram Positive Identification (GP ID) system for the identification of Beta Hemolytic Streptococci (BHS). Materials and Methods: A total 148 BHS which were isolated from various clinical specimens were included in the study. Isolates were identified by Bruker Biotyper 3.4 and VITEK2 GP ID coupled to the agglutination test. Identification results for Bruker Biotyper 3.4 and VITEK2 GP ID systems were classified in three different categories as follows: correct identification, low level of discrimination, no identification/misidentification. Identification results of BHS were evaluated according to these categories. Results: BHS isolates were defined as Streptococcus pyogenes (n: 69), Streptococcus agalactiae (n: 59), Streptococcus dysgalactiae serogroup C (n: 5), and Streptococcus dysgalactiae serogroup G (n: 15) according to latex agglutination assay. MALDI-TOF MS analysis of 148 BHS isolates yielded correct identification for 89.9% of S. pyogenes , 64.4% of S. agalactiae , and 95.0% S. dysagalactiae serogroup C and G; while VITEK2 GP ID gave correct identification for 62.3% of S. pyogenes , 67.8% of S. agalactiae , and 65.0% of S. dysagalactiae serogroup C and G. Conclusions: MALDI-TOF MS-based identification provides faster and more accurate identification of beta-hemolytic streptococcal species than VITEK2 GP ID system.

PubMedID: 25971934 Dermatophytes can invade the stratum corneum of the skin and other keratinized tissues and are responsible for a broad diversity of diseases of skin, nails and hair. Although the standard identification of dermatophytoses depends on macroscopic and microscopic characterization of the colonies grown on special media, there are a number of limitations owing to intraspecies morphological variability, atypical morphology or interspecies morphological similarity which entails improvement in the identification methods. Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method which proved to be effective for rapid and reliable identification of dermatophytes grown in cultures when compared to conventional methods. We evaluated the performance of Bruker MALDI-TOF MS System (Bruker Daltonics, Germany) for identification of clinically relevant dermatophytes. In order to increase the identification capacity of the system, we created supplemental spectral database entries using ten reference dermatophyte strains (ten species in two genera). The utility of the generated database was then challenged using a total of 126 dermatophytes (115 clinical isolates and 11 additional reference strains). The results were evaluated by both manufacturer-recommended and lowered cutoff scores. MALDI-TOF MS provided correct identification in 122 (96.8 %) and 113 (89.7 %) of the isolates with the lowered scores and using the supplemented database, respectively, versus 65 (51.6 %) and 17 (13.5 %) correct identifications obtained by the unmodified database and recommended scores at the genus and species levels, respectively. Our results support the potential utility of MALDI-TOF MS as a routine tool for accurate and reliable identification of dermatophytes. © 2015, Springer Science+Business Media Dordrecht.

Introduction: Staphylococcus aureus is one of the first bacteria colonizing in cystic fibrosis (CF) respiratory tract and different virulence factors are responsible for disease progression. It is not clear if CF S. aureus strains are more virulent than strains isolated from non-CF patients. Methodology: Biofilm production was detected by a modified tissue culture plate method, presence of genes encoding for Panton-Valentine leukocidin (PVL) was investigated by a signal amplified sandwich hybridization assay and antimicrobial susceptibility patterns were detected by disk diffusion method. Results: Staphylococcus aureus clinical isolates (n = 88) recovered from respiratory tract specimens in which 31 of them were from cystic fibrosis (CF) patients were analysed. Biofilm production was detected in 96.8% of CF isolates in which 32.3% exhibited strong positive phenotype and in 47.4% of non-CF isolates in which strong positive phenotype was not observed (p < 0.05). All CF isolates were methicillin susceptible, whereas 53.4% of non-CF isolates (n = 31) were methicillin resistant. No resistance was observed for vancomycin, chloramphenicol and trimethoprim/sulfamethoxazole in any of the isolates. PVL genes were detected only in two isolates (2.3%), one from each group, CF and non-CF, which both were methicillin susceptible Conclusion: Biofilm rather than PVL production appears to be an important virulence factor in CF patients.

Most of the clinically important bacteria causing infections in humans are capable of exhibit‐ ing resistance to antimicrobial agents commonly used for the treatment. Therefore, upon iso‐ lation of the organism in the clinical microbiology laboratory, characterization frequently also employs tests to detect its antimicrobial susceptibility. Thus, the report produced by clinical microbiology laboratory for the physician, also includes organism’s susceptibility profile to different antimicrobials along with its identification [1]. Antimicrobial susceptibility testing (AST) is performed on bacteria that are isolated from clinical specimens to determine if the bacterial etiology of concern can be killed or inhibited by antimicrobial drugs that are poten‐ tial choices for therapy, at the concentrations of the drugs that are attainable at the site of infec‐ tion using the dosing regimen indicated in the drug product’s labeling. The results of AST are generally reported with interpretive categories. The category “susceptible” indicates that the bacteria are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used. The “intermediate” category de‐ fines the bacteria for which the response rates to usually attainable blood and tissue levels of antimicrobial agent are lower compared to susceptible isolates. The intermediate category plays the role of a buffer zone between the susceptible and resistant categories, but also indi‐ cates a number of other possibilities; the antimicrobials which are concentrated at the site of infection may be regarded as options for treatment (e.g., nitrofurantoin for the urinary tract infections). The “resistant” category, however, defines the bacteria which are not inhibited by the usually achievable concentrations of the agent with normal dosage regimens and that the clinical efficacy of the agent against the isolate may not be sufficient [2]. Clinicians consider these interpretations to determine which antimicrobial agent might be effective in treating the particular patient. The primary role of routine microbiology laboratories is to provide accu‐

Pseudomonas aeruginosa is an opportunistic pathogen causing severe respiratory infections. The pathogenesis of these infections is multifactorial and the production of many virulence factors is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. The two well defined QS systems in P. aeruginosa, the las and rhl systems, rely on N-acyl homoserine lactone signal molecules, also termed autoinducers. We assessed the activity of QS-dependent virulence factors (including elastase, alkaline protease, pyocyanin and biofilm production) in respiratory isolates of P. aeruginosa and their relationship with antimicrobial susceptibility. We identified sixteen isolates displaying impaired phenotypic activity; among them, eleven isolates were also defective in autoinducer production, and therefore considered QS-deficient. Six of the QS-deficient isolates failed to amplify one or more of the four QS regulatory genes (lasI, lasR, rhlI, rhlR) with PCR: one isolate was negative for rhlR, two isolates were negative for rhlI and rhlR and three isolates were negative for all four genes. The isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials and statistically significant correlations were observed between the lack of elastase production and resistance to piperacillin and ceftazidime; between failure in alkaline protease production and resistance to tobramycin, piperacillin, piperacillin-tazobactam, cefepime, imipenem and ciprofloxacin; and between failure in pyocyanin production and resistance to amikacin, tobramycin, ceftazidime, ciprofloxacin and ofloxacin. The results obtained indicate that, despite the pivotal role of QS in the pathogenesis of P. aeruginosa respiratory infections, QS-deficient strains are still capable of causing infections and tend to be less susceptible to antimicrobials.