Genotyping by Random Amplified Polymorphic DNA Assay of Acinetobacter baumannii Isolated from Blood Culture of Patients with Hematological Malignancies

Relevance. Acinetobacter baumannii is a significant nosocomial pathogen that can cause severe infections, especially in immunocompromised patients. Aims. This study aimed to investigate clonal diversity of A. baumannii isolated from blood culture in hematological patients by random amplified polymorphic DNA assay (RAPD). Materials & Methods. Genotyping of A.baumannii isolated from blood culture in hematological patients in 7 Russian hospitals (2003–2017) was assessed by RAPD-PCR with primer OPA-2 (5’-TGCCGAGCTG-3’). The computer-assisted analysis was performed by using GelJ software by UPGMA method and Dice similarity coefficient for banding patterns comparison. Using a similarity coefficient (SC) of ≥ 65%, the strains were grouped. Based on the similarity coefficient, the strains were determined as genetically related (≥ 80%). Strains had identical RAPD-patterns if the similarity coefficient was 100%. Results. A total of 96 A. baumannii strains were examined, of those 77 (80.2%) were nonsusceptible to carbapenems. Acquired OXA-carbapenemase genes were detected among 79.2% carbapenem non-susceptible strains. RAPD-PCR genotyping revealed 84 RAPD patterns. The four groups (A-D) including 98% strains were defined by similarity coefficient ≥65%. The predominant group A included 58 (60.4%) strains, the C and B groups – had 15 strains (15.6%) each, and the group D – 6 strains (6.3%). A total of 82 (85.4%) genetically related A. baumannii with a similarity coefficient ≥ of 80% were allocated into 20 clusters. Identical RAPD-patterns were defined for 22 strains that belonged to 6 clusters within the group A and 1 cluster within the group B. Strains with identical RAPD-patterns were detected in a single hospital as well as in several hospitals located in different cities. Conclusions. The current study has demonstrated genetic diversity and clonal dissemination of A. baumannii in hematological departments

Molecular characterization of extended-spectrum β-lactamase-producing <i>Escherichia coli</i> collected from patients with hematological malignancies during chemotherapy cycles

Objective: to evaluate the genetic relatedness of extended-spectrum β-lactamase (ESBL) producing Escherichia coli isolated from the gut in patients with acute myeloid leukemia and lymphoma at admission and during chemotherapy cycles. Materials and methods. The prospective study (2013–2014) included 73 patients (median age 39 years) with acute myeloid leukemia (n = 25) and lymphoma (n = 48). The follow-up period lasted for 96 days. ESBL-producing E. coli isolated from the gut were included in this study. ESBL-production was confirmed by phenotypic tests, blaCTX-M and blaTEM genes were detected by polymerase chain reaction, and genotyping was performed by ERIC (Enterobacterial Repetitive Intergenic Consensus) polymerase chain reaction. Results. ESBL-producing E. coli were detected in 39 (53 %) of 73 patients: of them 12 (16 %) patients were colonized at admission and 27 (37 %) patients – during chemotherapy cycles. Gene blaCTX-M was detected in 67 % of E. coli, blaTEM – in 41 %, both genes – in 26 %. There was no genetically related ESBL-producing E. coli among 12 isolates detected at admission. Genetic relatedness was detected in 16 (59 %) of 27 isolates obtained during a hospital stay. Genetically related ESBL-producing E. coli were isolated from patients hospitalized in the same and different departments, these isolates were characterized by the presence of both identical and various determinants of resistance. Conclusion. Our data demonstrated the possibility of patient-to-patient transmission of ESBL-producing E. coli isolated from the gut during a hospital stay

Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM)

Observation of Cytotoxicity of Phosphonium Derivatives Is Explained: Metabolism Inhibition and Adhesion Alteration

The search for new antibiotics, substances that kill prokaryotic cells and do not kill eukaryotic cells, is an urgent need for modern medicine. Among the most promising are derivatives of triphenylphosphonium, which can protect the infected organs of mammals and heal damaged cells as mitochondria-targeted antioxidants. In addition to the antioxidant action, triphenylphosphonium derivatives exhibit antibacterial activity. It has recently been reported that triphenylphosphonium derivatives cause either cytotoxic effects or inhibition of cellular metabolism at submicromolar concentrations. In this work, we analyzed the MTT data using microscopy and compared them with data on changes in the luminescence of bacteria. We have shown that, at submicromolar concentrations, only metabolism is inhibited, while an increase in alkyltriphenylphosphonium (CnTPP) concentration leads to adhesion alteration. Thus, our data on eukaryotic and prokaryotic cells confirm a decrease in the metabolic activity of cells by CnTPPs but do not confirm a cytocidal effect of TPPs at submicromolar concentrations. This allows us to consider CnTPP as a non-toxic antibacterial drug at low concentrations and a relatively safe vector for delivering other antibacterial substances into bacterial cells

Antimicrobial resistance of <i>Enterococcus faecium</i> and <i>Enterococcus faecalis</i>, isolated from blood culture of patients with hematological malignancies during different study periods

Objective: to determine antimicrobial resistance of Enterococcus faecium and Enterococcus faecalis isolated from blood culture of hematological patients during different study periods.Materials and methods. Antimicrobial susceptibility of Enterococcus spp., collected as part of the multicenter study was tested by the broth microdilution method (USA Clinical and Laboratory Standards Institute (CLSI), 2018), to daptomycin by Etest (bioMeriéux, France). High-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR) was performed by the agar dilution method (CLSI (Oxoid, UK), 2018).Results. The susceptibility of 366 E. faecium (157 in 2002-2009 and 209 in 2010-2017) and 86 E. faecalis (44 in 20022009 and 42 in 2010-2017) was studied. In the second study period (2010-2017) the rise of vancomycin-resistant E. faecium (VREF) increased from 8.3 % to 23.4 % (p = 0.0001), and two linezolid-resistant (LREF) were identified. All VREF and LREF remained susceptible to daptomycin and tigecycline. The rate of susceptible to tetracycline E. faecium remained the same (73.9 and 74.6 %), and an increase in susceptibility to chloramphenicol (74.5 and 82.3 %) was observed. Susceptibility of E. faecium to tetracycline was detected with almost the same rate and in a part of isolates, the increase of susceptibility to chloramphenicol was registered during the analyzed periods. The rise of E. faecium susceptible to HLGR and HLSR has increased significantly in 2010-2017 compared to 2002-2009. Erythromycin, levofloxacin, ampicillin and penicillin had the least activity against E. faecium (less than 5 %).All E. faecalis were susceptible to tigecycline, linezolid, and teicoplanin. Only one of E. faecalis had intermediate resistance to vancomycin. High susceptibility to ampicillin in E. faecalis remained unchanged (97.7 and 97.6 %, respectively). In the second period of the study the rise of susceptible E. faecalis decreased significantly to penicillin (from 97.7 % to 76.2 %), to levofloxacin (from 59.1 % to 31 %), to HLSR (from 52.3 % до 31 %), and to HLGR (from 47.7 % to 26.2 %), remained unchanged to chloramphenicol (52.3 % and 50 %) and was minimal to erythromycin and tetracycline.Conclusion. The study demonstrated higher rates of antibiotic resistance among E. faecium, which consisted of an increase in VREF and the appearance of linezolid-resistant strains. High susceptibility to ampicillin remained in E. faecalis, but there was an increase in resistance to penicillin and aminoglycosides

Comparative Analysis of the lux Operons in Aliivibrio logei KCh1 (a Kamchatka Isolate) and Aliivibrio salmonicida ▿

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as Aliivibrio logei. We sequenced the entire lux operon of A. logei KCh1 and showed that it is substantially similar to the lux operon of Aliivibrio salmonicida. It was demonstrated that the reduced production of bioluminescence in A. salmonicida is most likely defined by a specific defect in its luxD gene

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2′-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria