The aim of the present study was to characterize sporadic cases and an outbreak of NDM-like-producing Enterobacteriaceae recovered from hospital settings, in Czechia. During 2016, 18 Entrobacteriaceae isolates including 10 Enterobacter cloacae complex (9 E. xiangfangensis and 1 E. asburiae), 4 Escherichia coli, 1 Kluyvera intermedia, 1 Klebsiella pneumoniae, 1 Klebsiella oxytoca, and 1 Raoultella ornithinolytica that produced NDM-like carbapenemases were isolated from 15 patients. Three of the patients were colonized or infected by two different NDM-like producers. Moreover, an NDM-4-producing isolate of E. cloacae complex, isolated in 2012, was studied for comparative purposes. All isolates of E. cloacae complex, except the E. asburiae, recovered from the same hospital, were assigned to ST182. Additionally, two E. coli belonged to ST167, while the remaining isolates were not clonally related. Thirteen isolates carried blaNDM−4, while six isolates carried blaNDM−1 (n = 3) or blaNDM−5 (n = 3). Almost all isolates carried blaNDM-like-carrying plasmids being positive for the IncX3 allele, except ST58 E. coli and ST14 K. pneumoniae isolates producing NDM-1. Analysis of plasmid sequences revealed that all IncX3 blaNDM-like-carrying plasmids exhibited a high similarity to each other and to previously described plasmids, like pNDM-QD28, reported from worldwide. However, NDM-4-encoding plasmids differed from other IncX3 plasmids by the insertion of a Tn3-like transposon. On the other hand, the ST58 E. coli and ST14 K. pneumoniae isolates carried two novel NDM-1-encoding plasmids, pKpn-35963cz, and pEsco-36073cz. Plasmid pKpn-35963cz that was an IncFIB(K) molecule contained an acquired sequence, encoding NDM-1 metallo-β-lactamase (MβL), which exhibited high similarity to the mosaic region of pS-3002cz from an ST11 K. pneumoniae from Czechia. Finally, pEsco-36073cz was a multireplicon A/C2+R NDM-1-encoding plasmid. Similar to other type 1 A/C2 plasmids, the blaNDM−1 gene was located within the ARI-A resistance island. These findings underlined that IncX3 plasmids have played a major role in the dissemination of blaNDM-like genes in Czech hospitals. In combination with further evolvement of NDM-like-encoding MDR plasmids through reshuffling, NDM-like producers pose an important public threat.

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Wild corvids were examined for presence of carbapenemase-producing Gram-negative bacteria in the USA. A total of thirteen isolates were detected among 590 faecal samples of American crow; eleven Providencia rettgeri isolates harbouring blaIMP-27 on the chromosome as a class 2 integron gene cassette within Tn7 transposon, one isolate of Klebsiella pneumoniae ST258 carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and one isolate of Enterobacter bugandensis with blaIMI-1 located within EcloIMEX-2.

The resistance to carbapenems is usually mediated by enzymes hydrolyzing β-lactam ring. Recently, an alternative way of the modification of the antibiotic, a β-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC–MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived β-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), β-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated β-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC–MS, we were able to identify meropenem-derived β-lactone directly according to the different retention time. All strains with a predominant β-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived β-lactone. We also identified β-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC–MS to detect meropenem-derived β-lactone.

Цель исследования: изучить механизмы формирования и тромбодинамические свойства кровяного сгустка при тромбообразовании in vitro и представить сравнительное исследование кинетики тромбообразования с помощью реологических и электрореологических методов в условиях постоянного и осциллирующего сдвигового вискозиметрического течения; оценить свертывание цельной и консервированной крови, ее вязкоупругие свой ства и влияние фибриногена и декстранов на формирование сгустка. Материалы и методы. Вязкость и удельную электропроводимость нормальной и консервированной с CPD-A1-адениновым раствором крови измеряли ротационным вискозиметром Low Shear 30 Contraves (LS30) с коаксиальными цилиндрами и копией измерительной системы MS1/1 со встроенными электродами (разработана на базе LS30) в условиях постоянного сдвигового потока и в электрическом поле. Вязкоупругие свойства нормальной цельной крови исследовали с помощью реометра Physica MCR 301 (Anton Paar, Austria) в условиях осциллирующего синусоидального потока. Результаты. Представлены результаты кинетики динамической вязкости и удельной электропроводимости консервированной крови в процессе коагуляции в условиях постоянного сдвигового потока и в электрическом поле. Исследованы вязкоупругие свойства цельной крови в условиях синусоидального осциллирующего вискозиметрического течения и представлены зависимости упругого модуля G' (storage modulus) и модуля потерь G'' (loss modulus), а также нормальных сил как функция времени при свертывании. Приводятся данные эффекта фибриногена и декстрана на упругий модуль G' и на модуль потерь G'' цельной крови в процессе коагуляции. Заключение. В условиях вискозиметрического течения при низких скоростях сдвига кинетика тромбообразования характеризуется начальным этапом постепенного увеличения кажущейся вязкости и уменьшения удельной электропроводимости крови и периодом интенсивной коагуляции, характеризирующимся экспоненциальным ростом вязкости и параллельным уменьшением удельной электропроводимости исследуемого образца. Установлено, что коагулирующая цельная кровь обладает нелинейными вязкоупругими свойствами. Кинетика образования сгустка в условиях осциллирующего вискозиметрического течения крови характеризуется увеличением упругого модуля G' и модуля потерь G'' со временем. Одновременно зарегистрирована и отрицательная нормальная сила исследуемого образца в зазоре между пластинами при постоянной толщине зазора. Повышение содержание фибриногена ускоряет коагуляцию и увеличивает значения упругого модуля G' и модуля потерь G'', а также и нормальной силы коагулирующей цельной крови. Objectives: to study the mechanisms of thrombus formation and thrombodynamic properties of a blood clot during coagulation in vitro and to present a comparative study of clot kinetics formation under conditions of steady and oscillating shear viscometric flow and at electric field by rheological and electrorheological methods; to evaluate the coagulation of whole and preserved blood, its viscoelastic properties and the effect of fi brinogen and dextrans on clot formation. Materials/ Methods. The viscosity and electrical conductivity of normal blood and blood preserved with CPD-A1-adenine solution were measured with a Low Shear 30 Contraves (LS30) rotational viscometer with coaxial cylinders and with a copy of the measuring system MS1 / 1 with builtin electrodes at a steady viscometric shear flow and in an electric field too. The viscoelastic properties of normal whole blood were investigated using a Physica MCR 301 rheometer (Anton Paar, Austria) at an oscillating sinusoidal flow. Results. Kinetics of the dynamic viscosity and electrical conductivity of preserved blood during coagulation at a steady shear flow and at electric field were obtained. The viscoelastic properties of whole blood were investigated under conditions of sinusoidal oscillating viscometric flow. The dependences of the elastic (storage) modulus G' and the loss modulus G'', as well as the normal coagulation forces as a function of time are presented. The effect of fibrinogen and dextran on the elastic modulus G' and the loss modulus G'' of whole blood during coagulation are presented. Conclusions. The kinetics of blood coagulation at low shear rates is characterized by a gradual increase of the apparent viscosity and a decrease in blood conductivity at the initial stage and an exponential increase in viscosity during intensive coagulation and a parallel decrease of conductivity too. It was established that coagulating whole blood exhibit nonlinear viscoelastic properties under conditions of sinusoidal blood flow. The kinetics of thrombus formation is characterized by increasing the elastic modulus G' (storage modulus) and the loss modulus G'' with time. The negative normal force in the gap between the plates is also registered at a constant thickness of the gap. An increase in fibrinogen content accelerates coagulation and increases the values of elastic modulus G' and loss modulus G', as well as of the normal force of coagulating whole blood.