An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

Molecular Epidemiology of Bulgarian Clinically Significant Staphylococcus aureus Isolates.

Severe infections due to highly virulent and resistant Staphylococcus aureus pose a serious health threat in Bulgaria and worldwide. The purpose of this study was to explore the clonal spread of recent clinically significant methicillin-susceptible S. aureus (MSSA) isolates from inpatients and outpatients treated in three university hospitals in Sofia, Bulgaria, during the period 2016-2020 and evaluate the relationship between their molecular epidemiology, virulence profiling, and antimicrobial resistance. A total of 85 isolates (invasive and noninvasive) were studied using RAPD analysis. Ten major clusters (A-K) were identified. The first major cluster A (31.8%) was found to be predominant during 2016 and 2017 and was widespread in two hospitals, unlike its case in the following years, when it was found to be replaced by newer cluster groups. All MSSA members of the second most common cluster F (11.8%) were recovered from the Military Medical Academy, mainly during 2018-2020, and were determined to be susceptible to all other groups of antimicrobials, except for penicillins without inhibitors because they harboured the blaZ gene. The newer cluster I, with 9.4% of the isolates absent in 2016-2017, showed significantly higher virulence and macrolide resistance (42.9%) due to ermB and ermC. All the isolated MSSA in groups F and I were nosocomial and mostly invasive. In conclusion, this 5-year study demonstrates the molecular epidemiology of MSSA infections in three Bulgarian hospitals. Findings can be helpful for the understanding of staphylococcal infection distribution in hospital settings and their prevention.

Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates.

Background: Severe infections of virulent methicillin-resistant Staphylococcus aureus (MRSA) are a serious health problem. The present study aimed to investigate clonal spread, virulence and antimicrobial resistance rates of Bulgarian MRSA isolates in 2016-2020. Methods: Molecular identification and mecA gene detection were performed with PCR. Clonal relatedness was evaluated by RAPD PCR and MLST. MRSA epidemiology, virulence and resistance patterns were investigated by PCR. Results: All 27 isolates were identified as S. aureus and were mecA positive, and all were susceptible to linezolid, tigecycline and vancomycin. The toxin genes hlg (in 92.6% of isolates), seb (77.8%), sei (77.8%), seh (59.3%), sej (55.6%), and seg (48.1%), were frequently found among the isolates. Epidemiological typing by RAPD identified 4 clones (16 isolates) and 11 were with a unique profile. MLST analysis of the same MRSA isolates showed five MLST clonal complexes and 11 ST types, including CC5 (33.3%) (ST5, ST221, ST4776), CC8 (22.2%) (ST8, ST239, ST72), CC15 (ST582), CC22 (14.8%) (ST217, ST5417), CC30 (ST30) CC398 (ST398), and CC59 (ST59). The isolates from CC5 showed higher virulence potential and almost all were macrolide resistant (ermB or ermC positive). CC8 isolates showed higher level of resistance. Conclusion: To the best of our knowledge, this study is the first describing the clonal spreading of Bulgarian MRSA and the association with their virulence and resistance determinants. Monitoring of MRSA epidemiology, resistance and virulence profile can lead to better prevention and faster therapeutic choice in cases of severe infections.

Spectrum of Causative Pathogens and Resistance Rates to Antibacterial Agents in Bacterial Prostatitis.

OBJECTIVE: To evaluate spectrum and resistance rates to antibacterial agents in causative pathogens of bacterial prostatitis in patients from Southern Europe, the Middle East, and Africa. MATERIALS: 1027 isolates from cultures of urine or expressed prostatic secretion, post-massage urine or seminal fluid, or urethral samples were considered. RESULTS: Escherichia coli (32%) and Enterococcus spp. (21%) were the most common isolates. Other Gram-negative, Gram-positive, and atypical pathogens accounted for 22%, 20%, and 5%, respectively. Resistance was <15% for piperacillin/tazobactam and carbapenems (both Gram-negative and -positive pathogens); <5% for glycopeptides against Gram-positive; 7%, 14%, and 20% for aminoglycosides, fosfomycin, and macrolides against Gram-negative pathogens, respectively; 10% for amoxicillin/clavulanate against Gram-positive pathogens; <20% for cephalosporins and fluoroquinolones against to Gram-negative pathogens (higher against Gram-positive pathogens); none for macrolides against atypical pathogens, but 20% and 27% for fluoroquinolones and tetracyclines. In West Africa, the resistance rates were generally higher, although the highest rates for ampicillin, cephalosporins, and fluoroquinolones were observed in the Gulf area. Lower rates were observed in Southeastern Europe. CONCLUSIONS: Resistance to antibiotics is a health problem requiring local health authorities to combat this phenomenon. Knowledge of the spectrum of pathogens and antibiotic resistance rates is crucial to assess local guidelines for the treatment of prostatitis.

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different ß-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes blaOXA-23 or blaOXA-72; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Isolation of Acinetobacter radioresistens from a clinical sample in Bulgaria.

We report on the role of Acinetobacter radioresistens in a case of pneumonia in an elderly patient and describe the challenge of correct identification of this species. A tracheobronchial culture taken from a patient in a Bulgarian hospital yielded a pure culture of Gram-negative, lactose-non-fermenting bacilli on MacConkey agar. Genus and species identification was performed by biochemical tests and sequencing of the rpoB gene. Antimicrobial susceptibility testing and screening for blaOXA-like carbapenemase genes was done using microbroth dilution and PCR and sequencing, respectively. The bacillus growing on MacConkey agar was initially identified by biochemical tests as Acinetobacter baumannii complex. Sequencing of the rpoB gene finally identified A. radioresistens. The strain harboured the carbapenemase gene blaOXA-23 without insertion sequences upstream of this gene and was susceptible to imipenem and meropenem. In conclusion, detection of A. radioresistens remains a challenge for routine laboratory diagnostics without performance of molecular identification methods. Although A. radioresistens can be a causative agent of opportunistic infections, in the present case its involvement in the development of pneumonia is doubtful.

Seroprevalence of Crimean-Congo hemorrhagic fever in southeastern Bulgaria.

Crimean-Congo hemorrhagic fever (CCHF), which is endemic in Bulgaria, is caused by the Crimean-Congo hemorrhagic fever virus (CCHFV). The seroprevalence of CCHFV in southeastern Bulgaria was examined in this study. For this purpose, a total of 751 human blood samples were collected and examined by indirect immunofluorescence assay. In addition, a questionnaire was completed for every participant. Anti-CCHFV antibodies were detected in 3.20% (24/751) of the tested sera. None of the seropositive individuals had a history of CCHF. The results indicate that the proportion of positive findings increase with age. The significant risk factors for CCHFV infection are tick bites (18.85%, 23/122), livestock breeding (6.15%, 16/260), and residing in rural areas (6.20%, 21/339).

Outbreak caused by NDM-1- and RmtB-producing Escherichia coli in Bulgaria.

Twelve consecutive carbapenem-resistant Escherichia coli isolates were recovered from patients (infection or colonization) hospitalized between March and September 2012 in different units at a hospital in Bulgaria. They all produced the carbapenemase NDM-1 and the extended-spectrum-ß-lactamase CTX-M-15, together with the 16S rRNA methylase RmtB, conferring high-level resistance to all aminoglycosides. All those isolates were clonally related and belonged to the same sequence type, ST101. In addition to being the first to identify NDM-producing isolates in Bulgaria, this is the very first study reporting an outbreak of NDM-1-producing E. coli in the world.

Comparison of the prevalence of Crimean-Congo hemorrhagic fever virus in endemic and non-endemic Bulgarian locations.

BACKGROUND & OBJECTIVES: The Balkans is an endemic region for Crimean-Congo hemorrhagic fever (CCHF), caused by the CCHF virus (CCHFV). Several Bulgarian regions comprised of smaller locations are categorized either as endemic or non-endemic for CCHF. However, little is known about the dynamics that underlie the development of endemicity within the locations throughout the years. METHODS: Seven locations categorized as endemic in one central Bulgarian region (Stara Zagora) were compared to seven non-endemic areas. During the period 2006-12, a total of 1775 blood samples from cattle, were tested for anti-CCHFV antibodies using an indirect immunofluorescence antibody assay. Also, the infestation of 617 mature ticks for CCHFV was studied using a combination of an immunofluorescence haemocytes assay and molecular-virological methods. RESULTS: Anti-CCHFV antibodies were established in 7.89% (140/1775) of the sera. The average CCHFV-infestation in the ticks was 1.46% (9/617). CCHFV was detected in three tick species: H.m. marginatum (3.73%, 6/161), being the main vector of the infection; R. sanguineus (1.63%, 2/123); and I. ricinus (1.96%, 1/51). INTERPRETATION & CONCLUSION: The data for the endemic and non-endemic locations did not reveal significant differences for the prevalence of CCHFV. Mosaic dispersion of the virus was determined in the studied region and the results did not vary significantly throughout the investigated years.

Crimean-Congo hemorrhagic fever virus-tick survey in endemic areas in Bulgaria.

The Balkan Peninsula and Bulgaria in particular, is a well-known endemic region for Crimean-Congo hemorrhagic fever (CCHF). This study describes the prevalence of Crimean-Congo hemorrhagic fever virus (CCHFV) among tick populations from areas, previously recognized with emerging cases of CCHF disease in humans. These include regions from the Southeastern (regions of Kardzhali and Haskovo) and Central (region of Stara Zagora) parts of the country. For the period 2006-2010 a total of 911 adult ticks, collected from livestock in endemic areas were studied for presence of CCHFV by an immunofluorescence-hemocytes assay (IFHA) and a reverse transcription-polymerase chain reaction (RT-PCR). The detection rate of CCHFV in the tick population was 2.09%. The prevalence of the virus was determined between 2.01% and 4.83% in the regions of Kardzhali and Haskovo, respectively (Southeastern Bulgaria). In the Central part of the country CCHFV infestation of the ticks was observed in 1.46% (region of Stara Zagora). The results confirmed the mosaic dispersion of CCHFV in the investigated regions. The principal infection vector in the surveyed areas was confirmed to be Hyalomma marginatum marginatum. Rhipicephalus sanguineus and Ixodes ricinus were also detected and may play a role in the transmission of CCHFV. Species distribution of CCHFV-positive ticks was as follows: H. m. marginatum-4.93%; R. sanguineus-2.33%; I. ricinus-1.02%. The combination of IFHA and RT-PCR that are used in this study are useful tools in the algorithm for monitoring endemic areas in Bulgaria.

Development of a pyrosequencing assay for rapid assessment of quinolone resistance in Acinetobacter baumannii isolates.

Rapid and reliable assessment of Acinetobacter baumannii resistance to quinolones was successfully achieved through pyrosequencing of the gyrA and parC quinolone-resistance determining regions. A strong correlation was found between quinolone resistance and mutations in gyrA codon 83 and/or in the parC gene (codons 80 or 84). Absence of QRDR mutations was associated with susceptibility to quinolones.