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Objectives: Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs.Methods: Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 μg/mL) to the initial antibiotic incubation step.Results: The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%-90.55%) and 99.29% (95% CI: 96.11%-99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%-92.01%) and 80.85% (95% CI: 73.38%-86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%-90.78%) and 91.37% (95% CI: 85.41%-95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive.Conclusions: Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test. [ABSTRACT FROM AUTHOR]

Objectives: Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods: The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with theCIM and Carba NPtests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion: The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions: The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories. [ABSTRACT FROM AUTHOR]

Introduction. Resistance to carbapenems represents a public health across the world. Screening for carbapenem resistance can be done starting from the antibiogram, but confirmatory methods are usually performed in reference centers. Methods for rapid identification of carbapenemase activity are required to empower laboratories to detect carbapenemases and raise signals for infectious disease physicians, antibiotic stewardship teams and hospital hygiene specialists. Materials and methods. Eppendorf tubes containing Klebsiella pneumoniae strains with carbapenemase activity (KPC-1, NDM-1, OXA-48) and others with no carbapenemase activity (K. pneumoniae ATCC 700603 and E. coli ATCC 25922), as well as positive and negative controls were incubated at 37 °C with varying concentrations of Meropenem. After incubation, samples were spun down until a pellet formed and the supernatant was used to challenge an exponentially growing E. coli culture. This second culture was followed with nephelometric measurements every half hour. Experiments were carried out in three technical replicates. The statistical analysis was done with R (version 3.3.3) and graphs generated in RStudio (version 1.0.136) with ggplot2 (version 2.1.0). Mann Whitney U Test with the assumption of independent samples was used to analyse the results. Results. The doses of Meropenem were chosen after dose ranging experiments and multiple incubation times (from 30 minutes to two hours) were tested in order to evaluate the robustness of the method. In the cases where the antibiotic was first incubated with strains that had carbapenemase activity, the growth of Escherichia coli ATCC 25 922 was non-hindered, matching that of controls. In the absence of carbapenemase activity and in the case of negative controls, the growth indicator strain was inhibited. Statistically significant differences were identified for 0.3, as well as a 0.5 McFarland index as early as 1.5 hours total work time. Conclusions. The discovery of carbapenem resistance has direct clinical, as well as epidemiological implications. Among the mechanisms implicated in resistance to carbapenems, the presence of a carbapenemase is one of the most worrying from an epidemiological standpoint. This new tool we describe herein can be performed in all clinical microbiology laboratories, with no extra expense (as opposed to CarbaNP and BlueCarba tests). [ABSTRACT FROM AUTHOR]

Carbapenems are last resort antibiotics used in infections caused by multidrug-resistant bacteria. Carbapenem-resistant Enterobacteriaceae infections are an increasingly global threat, especially because carbapenemase production is more frequent than other resistance mechanisms and is encoded by genes located on transferable genetic structures. The production of classes A (KPC), B (IMP, VIM, NDM) and D (OXA-48 and its variants) of carbapenemases has important clinical consequences and is a constant pool of resistance. This review tries to show the diversity of carbapenemases and to underline their importance. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) represents a growing public health problem worldwide. Infections with such bacteria lead to longer hospitalization times, higher healthcare costs and greater morbidity and mortality. Thus, there is a greater need for rapid detection methods in order to limit their spread. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) are a series of epidemiologically-important microorganisms of great concern due to their high levels of resistance. This review aimed to update the background information on the ESKAPE pathogens as well as to provide a summary of the numerous phenotypic and molecular methods used to detect their AMR mechanisms. While they are usually linked to hospital acquired infections, AMR is also spreading in the veterinary and the environmental sectors. Yet, the epidemiological loop closes with patients which, when infected with such pathogens, often lack therapeutic options. Thus, it was aimed to give the article a One Health perspective. [ABSTRACT FROM AUTHOR]

Introduction: Respiratory tract infections (upper and lower) represent one of the most frequent problems that lead to community medical visits, according to official data. In the hospital environment, the treatment of infections requiring inpatient care needs to be carried out in accordance with institutional guidelines, adapted to local antimicrobial resistance patterns. Information about the spectrum of bacterial resistance is often absent. Materials and methods: A retrospective study of the antibiogram registry of the Bacteriology Laboratory of the “Marius Nasta” National Institute of Pneumophtysiology (NIP) in Bucharest. The most frequent microorganisms isolated from pulmonary samples of adult inpatients in 2015 were analyzed. Results: The resistance of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus spp., Klebsiella spp., Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa was analyzed. We reported the total number of isolates, the number of analyzed isolates (the ones that come from patients who meet the inclusion criteria) and the absolute and proportion figures for resistant, intermediate and sensitive strains. Resistance is described for each of the microorganisms. Special cases of resistance (like methicillin resistant staphylococci and carbapenem resistant enterobacteriaceae; CRE) have been highlighted because of the need to monitor their evolution over time as well as the importance to approach them systemically (prevention and treatment). Discussions: The spectrum of resistance varies according to the ward where the patient was cared for. The isolates from the medical wards were, in general, less resistant than those from the ICU/surgery wards. Conclusions: Due to the high variability in the spectrum of bacterial resistance of the most frequent isolates from medical practice, heightened attention is required in choosing the appropriate empiric antibiotic coverage and adherence to sampling and diagnostic procedures. The present article helps to better characterize the epidemiology of infections and antibiotic resistance in the “Marius Nasta” Institute; it identifies the difficulties faced in gathering and analyzing the data and can be considered as a reference point to improve medical practice policies with regards to the use of antibiotics. [ABSTRACT FROM AUTHOR]

The production of carbapenemases is one of the most important mechanisms that lead to carbapenem resistance. At the same time, the carbapenemases present on extracromosomial genetic material (plasmids) are transmitted between various bacteria. Although the genes Klebsiella, Enterobacter and Escherichia are most commonly involved in infections, the identification of carbapenemases in other genes may be very important for controlling infections. In this article, we will review a recent study published in the Morbidity and Mortality Weekly Report, which evaluated the production of carbapenemases in enterobacteria belonging to genes less commonly involved in clinical practice. All strains of enterobacteria transmitted to the Center for Disease Control (CDC) in Atlanta and the Public Health Laboratory of the Minnesota Health Department (MDH) from 1 January 2014 to 30 September 2017 were screened for the identification of five carbapenemases (KPC, NDM, OXA-48, IMP, and VIM). Among these, the results for the genera Klebsiella, Enterobacter and Escherichia were excluded. CDC received 1039 EPC-RC isolates during the study, of which 63 (6.1%) of the infrequently identified genera. Over the same period, 149 (12%) of the 1241 CREs were producing carbapenemases. Although in small numbers, isolates from more infrequent genera of enterobacteria that produce carbapenemases may be an important reservoir for dissemination to other bacteria. Additionally, we will add suggestions for current, cost-effective methodological solutions that can improve the knowledge of carbapenemase production in our country. [ABSTRACT FROM AUTHOR]

Globally, more than 50% of HIV infected people are women. HIV infection in women has its particularities, especially because of pregnancy and because of the adverse effects of antiretroviral therapy. Heterosexual contact is the most frequent way of HIV transmission in women. Some gynecological infections like vaginal candidiasis seem to be more persistent and more severe as compared to uninfected female patients. Also, HIV infected women have a higher risk for cervical dysplasia and cancer as compared to uninfected women. The risk of mother-to-child transmission of the virus has dropped significantly in the last decades because of the implementation of routine HIV screening testing in pregnancy, counseling regarding prevention and the large access to antiretroviral treatment. Today, because of the diversity of the therapeutic options and using good prophylactic measures, HIV infected women can have a normal life. [ABSTRACT FROM AUTHOR]

Acute bronchitis is a frequent clinical entity, characterized by an acute, which is sometimes associated with upper respiratory tract infection. The m ajority of acute com m unity acquired bronchitis is caused by viral infections. Although in the absence of comorbidities and complications the prognosis is excellent, bronchitis is a frequent cause of antibiotic prescription. The present article wishes to review the clinical and functional aspects pertaining to the disease, as well as the microbiology and treatment of uncomplicated bronchitis in the immunocompetent adult. The role of influenza vaccination is highlighted as a preventive measure. We hope that the article will draw an alarm signal regarding antibiotic prescription in the frequent condition and that it will help in limiting the phenomenon of antibiotic resistance [ABSTRACT FROM AUTHOR]

Introduction: In a previously published article, we have presented aspects regarding the physiology of microcalorimetrie bacterial growth in a liquid broth medium, in the absence of continuous agitation. Metronidazole is an antibacterial and antiparasitic agent used for the treatment of infections with anaerobic microorganisms as well as some parasitic infestations. The present work presents the growth of an aerobic, facultative anaerobic reference Escherichia coli strain (ATCC 25922), in the presence of metronidazole. Materials and Methods: The inoculum studied was a 0.1 McFarland unit suspension, which was then, diluted 1/100. A Setaram microDSC 7 Evo differential scanning microcalorimeter was used to register bacterial growth patterns. A constant volume of bacterial inoculum was pipeted into Hastelloy cells. Different concentrations of antibiotic were prepared to the same volume with the use of Physiologic Saline (PS) and were added to the bacterial culture, before incubation, leading to a constant volume of air in the headspace of the cell. Hermetic seal was realized with the use of a silicon o-ring. The working efficient volume of the cell was 0.94 ml. The total working bacteria-drug suspension was 0.70 ml. Results: The modifications brought about by metronidazole at the level of the Escherichia coli growth curve appeared in a dose-dependent manner. The addition of metronidazole did not modify the total heat produced by the bacterial growth process (as objectified by the area under curve). Discussion: Through the comparison of the microcalorimetrie results obtained by other authors and the comparative analysis of the bacterial growth curves, shows the fact that metronidazole has a particular effect, one which is not seen in the interaction with other antibiotic classes. Conclusions: Metronidazole seems to have a characteristic effect on the microcalorimetrie growth curve of a bacterial population that is developing in liquid media, in oxygen restrictive conditions. [ABSTRACT FROM AUTHOR]

Bacterial resistance to antibiotics is a globally recognized healthcare problem. B-lactam resistance în Gram negative bacteria, mediated through mecanisms like ESBLs (extended spectrum beta-lactamases), cephalosporinases and carbapenemases, is one of the most important. This study aims to identify the most important tests which can be applied in the laboratory in order to correctly identify Gram-negative bacteria (Enterobacterales, Pseudomonas spp., Acidentobacter spp.) which are resistant to this class of antibiotics and their resistance mechanisms, apply them on the microorganisms isolated in a clinical hospital from Bucharest, Romania, describe and report the findings, followed by the proposal of measures that can reduce the burden of these bacteria and their resistance mechanisms. In order to support the national effort in the fight against the antimicrobial resistance phenomenon, a new phenotipic laboratory identification test is proposed, using resources available in Romania. [ABSTRACT FROM AUTHOR]

Introduction: Bacterial growth in liquid growth medium, in the absence of cell agitation subjects the microorganisms to different oxygenation regimes, due to limitation in oxygen diffusion rate. When oxygen itself is limited, microorganisms will grow in anaerobic and microaerophilic conditions. The goal of this paper is to present the differences in bacterial growth associated with variations in residual oxygen inside the microcalorimetric cells. Materials and Methods: The bacterial strain chosen for this investigation was Escherichia coli (ATCC 25922). The studied inoculum represents a hundred fold dilution of a 0.1 McFarland units suspension, subjected to stationary growth, lacking homogenization, at 37°C in a differential scanning Setaram microDSC III microcalorimeter. To explore the metabolism in the context of limited oxygen supply, the bacterial suspension was cultivated in Hastelloy cells, hermetically sealed with silicon o-rings, provided by the manufacturer. The total efficient volume of the cell is 0.94ml. The volume of residual air (and implicitly, of oxygen) in the headspace of the cell was adjusted by addition to the bacterial suspension of Physiological Saline (PS). Results: The comparison the growth curves obtained by recording the growth of 0.6 ml of suspension with those of 0.3 ml and 0.3 ml with addition of 0.3 ml PS shows the differences in bacterial metabolism when oxygen and nutritive medium are in different volumes. Reducing of the bacterial suspension by half resulted in decrease in the intensity of the first peak, while at the same time leading to increase in the second peak. The addition of PS maintained roughly the same first peak intensity, while bringing the second peak intensity to its original level. Discussions: Published studies of microbial microcalorimetry do not present data of base processes that lead to generation of the microcalorimetric curve. This first experimental analysis of bacterial growth helps in understanding the processes of growth under limited oxygen conditions. We propose that differences in remaining oxygen inside the microcalorimetric cells would, in part, be responsible for the variability seen in the specialty literature. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis (Nm) is the pathogen carried asymptomatically in the nasopharynx but which under certain conditions can produce meningitis or even multi-organ failure. Experimental induction of meningitis in an animal model is necessary for testing new nanopharmaceutical products. Thus, the aim of the study was to establish the concentration of Nm capable of inducing the disease. We carried out 2 studies where we tested different concentrations of Nm obtained by the nephelometric and spectrophotometric methods. In the first study we included 20 BALB/c mice, and in the second 24. Local and general clinical signs, complete with body temperature values, body weight, hematological, microbiological and histopathological examination were important indicators for the assessment of the establishment of meningitis. The clinical results have pronounced depression in the first 2 days after inoculation, then the general condition will appear. The microbiological and histopathological examination indicated the presence of bacteria at the brain level and specific meningeal lesions only in the case of the second attempt, but the early mortality, requires additional testing of the bacterial concentration that induces meningites in a stable and persistent way so that treatment against the condition can be tested later. [ABSTRACT FROM AUTHOR]

Background: Romania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania. Methods: The strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization. Results: Both clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly blaOXA-23, blaOXA-24, blaSHV, blaTEM and blaGES, while Pa strains blaIMP, blaVIM, blaNDM, blaVEB, blaGES and blaTEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for blaOXA-23 or blaOXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters. Conclusion: Our study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment. [ABSTRACT FROM AUTHOR]

Chronic infections represent an important burden on the healthcare system and have a significant impact on the patients' quality of life. While Staphylococcus spp. are commensal bacteria, they can become pathogenic, leading to various types of infections. In this study we aimed to characterize the virulence profiles of staphylococcal strains involved in difficult-to-treat skin and soft tissue infections, from both phenotypic and genotypic points of view. Phenotypic ability of the strains to secrete soluble virulence factors was assessed by a culturing dependent assay and their capacity to develop biofilms on inert substrate was screened by an adapted crystal violet microtiter method. We also tested the presence of several virulence genes by PCR. Most of the studied strains were isolated from purulent secretions of acne lesions and frequently secreted two or three soluble virulence factors. Most frequently secreted soluble virulence factors were caseinase (89%), lipase (71%) and lecithinase (67%). Almost half of the strains produced a well-represented biofilm. The molecular characterization showed the presence of the genes cna, hlg, clfA, and clfB. Staphylococcal strains that produce difficult-to-treat skin and soft tissue infections seem to be characterized by an enhanced ability to produce different soluble virulence factors and to develop biofilms in vitro. Further studies need to be developed in other Staphylococcus spp. infections in order to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

In the context of SARS-CoV-2 infection, the identification of a vaccine has become vital for stopping the pandemic. The consortium formed by the OncoGen Center and the Cantacuzino Institute (CI) evaluates a candidate vaccine whose vaccine component is represented by a combination of peptides specifically recognized by the 2 components of the adaptative immune system. Each new vaccine must undergo mandatory tests, even if they are currently accelerated or overlapping, with the consent of the European Medicines Agency (EMA). One of the tests relates to repeated dose toxicity. The aim was to setup a protocol for repeated dose toxicity testing in rats, the results of which would allow the transition to the clinical phase I dossier. The protocol was designed based on EMA guidelines for vaccine safety testing and rat-human translation. The proposed human dose, route of administration and repeat administration were taken into account. Clinical signs, immunological and biochemical parameters will be analysed. Histological examination will be performed on 34 organs. The protocol was approved by the ethics committee of the CI and authorized by the competent authority. In order to evaluate the repeated dose toxicity of a vaccine candidate against COVID-19, we propose a robust, replicable, effective and translational study protocol. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of m ethicillin-resistcmt Staphylococcus aureus has increased in the last decades. The result was the increase of mortality due to systemic infections thus representing an issue of public health. Purpose: Cuantification of Staphylococcus aureus and Streptococcus pneumoniae strains carrying antibiotic resistance isolated in the N ational Institute of Pneum ophtisiology "Marius Nasta", Bucharest Matherials and methods: The study is retrospective and covers a time period of one year. The study was elaborated by analysis of the microbiology laboratory database of the National Institute of Pneum ophtisiology "Marius Nasta", Bucharest. We have selected the results of the bacteriological tests regarding Staphylococcus aureus and Streptococcus pneumoniae identification and have analyzed the cases of infections with methicillinresistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. The samples included: sputum , alveolar lavage, pharyngeal exudate, pus etc. Results: Streptococcus pneumoniae was identified in 69 samples (sputum, aspirate, lavage). A percent of 59,42% (4 1 cases) proved to be penicillin-resistant. Staphylococcus aureus was identified in 167 samples of which 40.72% (68 cases) were m ethicillin-resistant. Conclusions: We wanted to highlight the importance of monitoring and reporting of bacterial resistance. The knowledge of the present situation is of a real help when it comes to correctly establishing the antibiotic use politics (deceasing the risk of inadequate use). [ABSTRACT FROM AUTHOR]

Intoduction: Oral and pulmonary Candida spp. infections are opportunistic infections. The most frequently affected are the immunosuppressed individuals suffering from HIV, tuberculosis, viral hepatitis infection or cancer. Materials and Methods: This retrospective study was conducted fora period of one year, in a hospital dedicated to infectious diseases. We selected from the database of the clinical microbiology laboratory positive sputum samples for any species of Candida spp., and then we selected the medical records of patients with positive samples diagnoses. Results: We identified 135 samples (aspirate and sputum) positive for Candida spp., from 123 patients. Of the total number of patients, 33 (26.8%) were diagnosed with oral candidiasis and 22 (17.9%) were diagnosed with Candida spp. pulmonary infection. Of the oral candidiasis group, 24 (72.7%) were diagnosed with HIV infection, 7 7 (33.3%) had tuberculosis, 11 (33,3%) had viral hepatitis and 6 (18.2%) had cancer. 7 7 (50%) had pulmonary candidiasis associated with other bacterial infections. 28 strains of Candida spp. were isolated from pulmonary samples, 13 (46,4%) being Candida albicans and 15 (53.6%) Candida non-albicans. Conclusions: Diagnosis and monitoring of the Candida spp. pulmonary infections represents an important issue. It helps to distinguish whether to use or not antifungal treatment considering that resistance to antifungals increased in the last decades. [ABSTRACT FROM AUTHOR]