The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.

The aim of this study was to determine the prevalence of type III effector proteins (ExoS, ExoU, ExoT and ExoY)-encoding genes among clonally unrelated nosocomial Pseudomonas aeruginosa strains and to analyze their distribution in respect to the infection site and antimicrobial resistance. Polymerase chain reaction-based detection of the genes was performed on 176 non-duplicate P. aeruginosa isolates from three University hospitals in Sofia, previously genotyped by random amplified polymorphic DNA technique. The prevalence of the studied genes was as follows: exoS-61.9%, exoU-32.4%, exoT-100%, and exoY-85.8%. The part of P. aeruginosa strains harboring either the exoS (54.0%) or the exoU (23.8%) gene was higher (P<0.001) than that of isolates containing both genes (8.5%). The gene dissemination varied according to the infection localization. The exoU gene manifested a higher spread (P<0.001) among multidrug-resistant (MDR) than in non-MDR strains (42.6 vs 18.7%). In conclusion, the P. aeruginosa type III secretion system is present in nearly all studied isolates but the individual isolates from distinct infection sites differ in their effector genotypes. The ubiquity of type III effector proteins-encoding genes among clinical isolates is consistent with an important role for this system in P. aeruginosa pathogenesis.

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In the Large Helical Device ͑LHD͒, various spectroscopic diagnostics have been applied to study the ablation process of an advanced impurity pellet, tracer-encapsulated solid pellet ͑TESPEL͒. The total light emission from the ablation cloud of TESPEL is measured by photomultipliers equipped with individual interference filters, which provide information about the TESPEL penetration depth. The spectra emitted from the TESPEL ablation cloud are measured with a 250 mm Czerny-Turner spectrometer equipped with an intensified charge coupled device detector, which is operated in the fast kinetic mode. This diagnostic allows us to evaluate the temporal evolution of the electron density in the TESPEL ablation cloud. In order to gain information about the spatial distribution of the cloud parameters, a nine image optical system that can simultaneously acquire nine images of the TESPEL ablation cloud has recently been developed. Several images of the TESPEL ablation cloud in different spectral domains will give us the spatial distribution of the TESPEL cloud density and temperature.

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/μL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species.

Our objective was to investigate the plasmid replicon-types involved in spread of ESBLs among Bulgarian Klebsiella pneumoniae and Escherichia coli. Sixty-three isolates, with transferable beta-lactam resistance determinants, collected between 2007 and 2009 in six medical institutions, were analysed with respect to their antimicrobial susceptibility, ESBL-, RAPD-, and plasmid replicon-type. Phylogenetic typing and screening for the O25b-ST131 lineage were carried out for E. coli. The predominant ESBLs were CTX-M-15 (81%) among E. coli and CTX-M-3 (58%) among K. pneumoniae. Other sporadically found ESBLs were SHV-12 and TEM-139, and for the first time in Bulgaria, CTX-M-1 and CTX-M-14. Replicon typing revealed that plasmids carrying blaCTX-M-3 exclusively belonged to IncL/M-type, while blaCTX-M-15 was predominantly (94%) associated with IncF-type plasmids. Among E. coli, 59% of the isolates were clonally related. Isolates of that cluster produced CTX-M-15, belonged to the O25b-ST131 lineage, predominantly harboured plasmids with the FIA replicon, and were found in five centres. Among CTX-M-3-producing K. pneumoniae, two prevailing RAPD-types were found, one remained restricted to one centre and the second was found in three centres. The incompatibility groups IncN and IncA/C linked with blaSHV-12 respectively blaTEM-139 were found only once. To the best of our knowledge, this is the first detailed investigation of plasmids carrying ESBL genes among Bulgarian isolates demonstrating wide distribution of conjugative IncF plasmids among CTX-M-15-producing E. coli and IncL/M plasmids among CTX-M-3 positive K. pneumoniae isolates.

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