Vasiliki Gennimata

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

ABSTRACT We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

Current perspectives on tigecycline resistance in Enterobacteriaceae: susceptibility testing issues and mechanisms of resistance

During the past decades, rates of multidrug-resistant (MDR) and carbapenem-resistant (CR) Enterobacteriaceae clinical isolates, mainly Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus spp. and Serratia marcescens, have increased, considerably restricting effective antimicrobial treatments. Tigecycline, the first member of the glycylcyclines, has been approved by the US Food and Drug Administration (FDA) for the treatment of complicated skin and soft-tissue, complicated intra-abdominal and community-acquired bacterial respiratory infections and is increasingly administered against MDR Enterobacteriaceae. Although resistance has gradually appeared, tigecycline still remains relatively active among Enterobacteriaceae, with resistance rates largely <10% in most wide-scale surveillance studies. Tigecycline resistance has been reported in some studies to be elevated among extended-spectrum β-lactamase (ESBL)-producing, MDR, extensively drug-resistant and CR isolates. Broth microdilution (BMD) is the reference method for tigecycline susceptibility testing, but disagreements have been reported between the methods applied for routine tigecycline susceptibility testing. Therefore, confirmation of daily tigecycline susceptibility testing with BMD appears important in order to avoid misclassification of isolates. Various mechanisms have been reported to confer tigecycline resistance, with RND-type transporters, mainly the AcrAB efflux pump, playing an important role. Other pumps and various control pathways are also implicated in tigecycline resistance. Overall, tigecycline is a potent therapeutic option for enterobacterial infections. Accurate detection of tigecycline susceptibility status and surveillance of resistant organisms in the hospital environment is necessary in order to optimise its use and to preserve tigecycline in our therapeutic arsenal.

In Vitro Activity of Tigecycline Against Acinetobacter baumannii: Global Epidemiology and Resistance Mechanisms

Acinetobacter baumannii is a pathogen of increasing concern, commonly causing outbreaks in the hospital environment. Of particular concern, A. baumannii strains exhibiting resistance to carbapenems, which were previously considered the treatment of choice for infected patients, have dramatically increased worldwide, leaving a few antibacterial choices. Tigecycline, a broad-spectrum modified minocycline derivative, isconsidered as a last resort drug against multidrug-resistant A. baumannii. Though, resistance to tigecycline has emerged and is growing notably following increasing tigecycline usage. Comparative evaluation of the tigecycline resistance rates reported worldwide is challenging due to the absence of official interpretative criteria for in vitro susceptibility testing and the discrepancies among the different susceptibility methodologies used, with broth microdilution being considered the reference method. Tigecycline resistance is mainly associated with resistance-nodulation-cell division (RND)-type transporters, mainly the AdeABC, AdeFGH and AdeIJK efflux pumps, but other resistance mechanisms have also been implicated. Tigecycline is still an attractive choice for A. baumannii, but further investigations are warranted so that treatment of MDR Α. baumannii could be guided by validated in vitro data.

The Role and Predictive Value of Cytokines in Atherosclerosis and Coronary Artery Disease

Atherosclerosis is currently regarded as a chronic inflammatory disease that is mediated by several types of cells and molecules. Emphasis has been placed on the role of cytokines and the way they act and interact to initiate and sustain inflammation in the microenvironment of an atherosclerotic plaque. Cytokines are invariably expressed by all cells involved in the pathogenesis of atherosclerosis, act on a variety of targets exerting multiple effects and are largely responsible for the crosstalk among endothelial, smooth muscle cells, leukocytes and other vascular residing cells. In the present paper our aim is to review current information on the role of the most commonly discussed cytokines in the process of atherogenesis and to discuss the prognostic significance of these cytokines in atherosclerosis and coronary artery disease.

Circulating Biomarkers Determining Inflammation in Atherosclerosis Progression.

Atherosclerosis is the main underlying pathology of cardiovascular disease and is precipitated by various hereditary and non-hereditary risk factors. Inflammation is considered an important step in the progression of atherosclerosis and involves numerous cells, mediators and cellular procedures. Therefore, a biomarker able to determine the vascular inflammatory status is imperative as the combination of inflammatory biomarkers with the classic risk factors might provide further information about atherosclerosis progression and cardiovascular risk. The identification of novel inflammatory molecules and the improvement in analytical methods allows the potential implementation of these tests in every day clinical practice. In the current article, we focus on the role of established and novel biomarkers in atherosclerosis progression and in the determination of cardiovascular risk. We also present recent data concerning the risk stratification of patients according to their inflammatory status and the possible anti-inflammatory treatment strategies.

Biomarkers in atrial fibrillation and heart failure.

Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in clinical practice and an important contributor to cardiovascular morbidity and mortality. Although the exact mechanisms behind AF are not completely elucidated, the underlying pathophysiological changes have been well described. Predisposal factors for AF include the older age, the increased left atrial size, the decreased left atrial function, the presence of heart failure and left ventricular systolic dysfunction and the presence of coronary heart disease or pulmonary or mitral valve disease. In addition to these factors, emerging evidence demonstrate that myocardial strain, fibrosis and inflammation, are associated with AF as well as the pathogenesis of the arrhythmia. The natruretic peptide system including Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP) is indicative of the level of myocardial strain which may predispose to AF. As a result, the aforementioned peptides are increased in AF patients. The levels of myocardial fibrosis biomarkers, such as ST2 and Galectin-3, are elevated suggesting atrial structural abnormalities, while the increased levels of CRP and Interleukin-6 supplement the inflammatory profile of AF patients. Emerging data for the aforementioned biomarkers are discussed in the present review.

Redox State in Atrial Fibrillation Pathogenesis and Relevant Therapeutic Approaches

BACKGROUND Myocardial redox state is a critical determinant of atrial biology, regulating cardiomyocyte apoptosis, ion channel function, and cardiac hypertrophy/fibrosis and function. Nevertheless, it remains unclear whether the targeting of atrial redox state is a rational therapeutic strategy for atrial fibrillation prevention. OBJECTIVE To review the role of atrial redox state and anti-oxidant therapies in atrial fibrillation. METHOD Published literature in Medline was searched for experimental and clinical evidence linking myocardial redox state with atrial fibrillation pathogenesis as well as studies looking into the role of redoxtargeting therapies in the prevention of atrial fibrillation. RESULTS Data from animal models have shown that altered myocardial nitroso-redox balance and NADPH oxidases activity are causally involved in the pathogenesis of atrial fibrillation. Similarly experimental animal data supports that increased reactive oxygen / nitrogen species formation in the atrial tissue is associated with altered electrophysiological properties of atrial myocytes and electrical remodeling, favoring atrial fibrillation development. In humans, randomized clinical studies using redox-related therapeutic approaches (e.g. statins or antioxidant agents) have not documented any benefits in the prevention of atrial fibrillation development (mainly post-operative atrial fibrillation risk). CONCLUSION Despite strong experimental and translational data supporting the role of atrial redox state in atrial fibrillation pathogenesis, such mechanistic evidence has not been translated to clinical benefits in atrial fibrillation risk in randomized clinical studies using redox-related therapies.

Staphylococcus aureus osteoarticular infections in children: an 8-year review of molecular microbiology, antibiotic resistance and clinical characteristics

Purpose. To investigate the clinical, phenotypic and genotypic characteristics of Staphylococcus aureus strains causing osteoarticular infections in a large paediatric series. Methodology. Medical records of children who were hospitalized with the diagnosis of community‐associated S. aureus (CA‐SA) osteomyelitis and/or septic arthritis in the two major tertiary paediatric hospitals of Athens during an 8‐year period (2007‐2015) were reviewed, and S. aureus isolates were analysed regarding antimicrobial resistance, detection of pathogenicity genes and genotyping using SCCmec, agr typing, PFGE and MLST. Results. During the study period, 123 children with CA‐SA osteoarticular infections were identified, and methicillin‐resistant S. aureus (MRSA) accounted for 44 of these (35.8 %). Children with MRSA infection had a significantly higher admission rate to the ICU (5.7 vs 0 %, P=0.04) and longer duration of hospitalization (21.6 vs 16.7 days, P=0.04). Sixty‐eight isolates [42 (methicillin‐sensitive S. aureus) MSSA and 26 MRSA] were available for molecular analysis. All MRSA strains were mecA‐positive and most carried the SCCmec IV cassette (23/26, 88 %) and belonged to the PFGE type C (24/26, 92.3 %), agr type 3 (24/26, 92.3 %) and the MLST ST80 clone (24/26, 92.3 %). In contrast, MSSA strains showed polyclonality by PFGE and agr typing. Regarding pathogenicity genes, MRSA vs MSSA isolates showed higher detection rates of PVL (96.2 vs 4.8 %, P<0.0001) and fib (80.8 vs 50 %, P=0.02). Conclusions. In our study a considerable number of S. aureus osteoarticular infections were due to CA‐MRSA isolates, most of which belonged to the ST80 clone and had a higher incidence of specific virulence factors, entailing higher ICU admission rates and a longer duration of hospitalization.

Medical and Social Aspects of Syphilis in the Balkans from the mid-19th Century to the Interwar.

Abstract The current study presents some aspects of syphilis in the Balkan Peninsula from the 19th century until the Interwar. Ever since the birth of modern Balkan States (Greece, Bulgaria, Turkey and Serbia), urbanization, poverty and the frequent wars have been considered the major factors conducive to the spread of syphilis. The measures against sex work and sexually transmitted diseases (STDs) were taken in two aspects, one medical and the other legislative. In this period, numerous hospitals for venereal diseases were established in the Balkan countries. In line with the international diagnostic approach and therapeutic standards, laboratory examinations in these Balkan hospitals included spirochete examination, Wassermann reaction, precipitation reaction and cerebrospinal fluid examination. Despite the strict legislation and the adoption of relevant laws against illegal sex work, public health services were unable to curb the spread of syphilis. Medical and social factors such as poverty, citizen’s ignorance of STDs, misguided medical perceptions, lack of sanitary control of prostitution and epidemiological studies, are highlighted in this study. These factors were the major causes that helped syphilis spread in the Balkan countries during the 19th and early 20th century. The value of these aspects as a historic paradigm is diachronic. Failure to comply with the laws and the dysfunction of public services during periods of war or socioeconomic crises are both factors facilitating the spread of STDs.

Reply to "Reliability of Gradient Diffusion Methods for Detection of Acquired Colistin Resistance".

We appreciate the interest of Nastro et al. in our recent article (1); their comments (2) offer an opportunity to discuss the work further. Our study was undertaken with a collection of Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates for which colistin (Cst) MICs were elevated. It was observed that among the commercial methods for Cst susceptibility testing (ST), gradient diffusion methods (Etest and MTS) produced high rates of very major errors (VME) in comparison with broth microdilution (BMD). Error rates for K. pneumoniae were estimated using EUCAST breakpoints, as susceptibility breakpoints do not exist in CLSI documents; their applicability for this species may be limited in countries employing CLSI interpretive criteria. As the automated system Vitek2 yielded no VME, we concluded that such systems may be preferable for routine colistin ST over gradient diffusion methods. We should note herein that neither Vitek2 nor gradient diffusion methods are U.S. FDA approved for the testing of colistin for clinical purposes. After reading thoroughly the letter of Nastro et al. (2), we still believe that our results are supported by the literature that they list. In response to the comments made by our colleagues, we point out the following. (i) Hindler and Humphries (3), applying the Etest to Cst-resistant Gram-negative bacteria, reported higher rates of VME (47 to 53%) than our study. (ii) Tan and Ng (4) recorded low rates of VME with Enterobacteriaceae, but their study included only 3 Cst-resistant Klebsiella spp. Of note, although detailed results per species were not presented, with the Etest, the MICs of 61% of Enterobacteriaceae were 1 to more than 2 log2 dilutions lower than those determined with the standard method. (iii) Behera et al. (5) tested only 6 polymyxin B-resistant K. pneumoniae and A. baumannii isolates, and of those isolates, 2 (33.3%) produced VME with the Etest in comparison with the reference BMD method; of interest, 2 of 4 isolates for which polymyxin B MICs were 8 to 16 g/ml gave VME by the Etest. (iv) Arroyo et al. (6) actually observed that the Etest misclassified as Cst susceptible a quite high proportion (9.1%) of the 22 A. baumannii isolates defined as resistant by BMD; the rate of 1.7% VME that Nastro et al. claim referred to the total of isolates (80.9% of those being Cst susceptible). (v) Lo-Ten-Foe et al. (7) did not report specific results, but they tested only 1 Cst-resistant A. baumannii isolate and 7 Cst-resistant K. pneumoniae isolates, and for all 8 isolates, the Cst MICs were very high ( 64 g/ml). VME are predictably limited for such MIC levels, and this was shown also in our original study (1), where gradient diffusion methods misidentified as susceptible very few isolates for which the Cst MIC was 32 g/ml. (vi) Maalej et al. (8) included only 6 Cst-resistant K. pneumoniae isolates. (vii) Lee et al. (9) tested no Cst-resistant A. baumannii isolates. All the aforementioned data indicate that, as with our findings, important disagreements occur between the results of gradient diffusion and dilution methods for isolates resistant to Cst, particularly those for which the MICs are close to the resistance breakpoints. Second, our collection actually included 35% of isolates for which Cst MIC values were at the resistance breakpoint; it is for such isolates that particular attention is required in clinical practice. The fact that other studies (3, 9), as claimed by Nastro et al., detected fewer isolates for which the MIC was 4 g/ml cannot imply clonality for our isolates, which were temporally and geographically diverse and derived from four distinct hospitals. Pulsed-field gel electrophoresis (PFGE) analysis that was performed previously detected at least 3 major clonal types for the A. baumannii and 4 types for the K. pneumoniae isolates. Third, evident heteroresistance was not detected in our study. It should be noted that all isolates in our study were incubated for 18 to 20 h, as is a common practice and was also the case in all studies that Nastro et al cited., which reported incubation times (3, 4, 6, 8). We should note that we compared the currently existing methods by following the available guidelines and aimed to draw conclusions applicable for clinical laboratories. It was not our intention to optimize the methodologies, and for that reason, we did not modify the recommended protocols. We agree that such an investigation would be important for the optimization of gradient diffusion methods. In conclusion, resistance to Cst among A. baumannii and K. pneumoniae strains is an alarming and unfortunately increasingly observed evolution not only in Greece but also in several European regions (10). Its accurate detection in clinical laboratories is extremely important for the appropriate treatment of the respective infections. Our study was prompted by notifications from hospital laboratories that gradient diffusion methods were producing much lower Cst MICs than automated systems. Those preliminary observations were validated in our study when ST results were compared with those of the reference BMD method and were supported by most studies that tested isolates for which Cst MICs were elevated. Overall, it is clearly implied that gradient diffusion methods have to be used with caution for Cst ST of multidrugresistant A. baumannii and K. pneumoniae isolates with potentially elevated Cst MICs, although their results may be of some