First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylo-coccus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enter-obacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cepha-losporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cepha-losporinase was the only beta-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices

Whole-Genome Sequencing-Based Resistome Analysis of Nosocomial Multidrug-Resistant Non-Fermenting Gram-Negative Pathogens from the Balkans

Non-fermenting Gram-negative bacilli (NFGNB), such as Pseudomonas aeruginosa and Acinetobacter baumannii, are among the major opportunistic pathogens involved in the global antibiotic resistance epidemic. They are designated as urgent/serious threats by the Centers for Disease Control and Prevention and are part of the World Health Organization’s list of critical priority pathogens. Also, Stenotrophomonas maltophilia is increasingly recognized as an emerging cause for healthcare-associated infections in intensive care units, life-threatening diseases in immunocompromised patients, and severe pulmonary infections in cystic fibrosis and COVID-19 individuals. The last annual report of the ECDC showed drastic differences in the proportions of NFGNB with resistance towards key antibiotics in different European Union/European Economic Area countries. The data for the Balkans are of particular concern, indicating more than 80% and 30% of invasive Acinetobacter spp. and P. aeruginosa isolates, respectively, to be carbapenem-resistant. Moreover, multidrug-resistant and extensively drug-resistant S. maltophilia from the region have been recently reported. The current situation in the Balkans includes a migrant crisis and reshaping of the Schengen Area border. This results in collision of diverse human populations subjected to different protocols for antimicrobial stewardship and infection control. The present review article summarizes the findings of whole-genome sequencing-based resistome analyses of nosocomial multidrug-resistant NFGNBs in the Balkan countries

Novel Microfluidics Device for Rapid Antibiotics Susceptibility Screening

In recent years, excessive utilization of antibiotics has led to the emergence of antibiotic microbial resistance on a planetary scale. This recent phenomenon represents a serious threat to public health, as well as an enormous burden for healthcare systems’ budgets worldwide. Novel, rapid and cheap methods for antibiotic susceptibility screening are urgently needed for this obstacle to be overcome. In this paper, we present a microfluidic device for on-chip antibiotic resistance testing, which allows for antibiotic microbial resistance detection within 6 hours. The design, fabrication and experimental utilization of the device are thoroughly described and analyzed, as well as possibilities for future automation of the whole process. The accessibility of such a device for all people, regardless of economic status, was of utmost importance for us during the development of the project

Study of the Bacterial, Fungal, and Archaeal Communities Structures near the Bulgarian Antarctic Research Base “St. Kliment Ohridski” on Livingston Island, Antarctica

As belonging to one of the most isolated continents on our planet, the microbial composition of different environments in Antarctica could hold a plethora of undiscovered species with the potential for biotechnological applications. This manuscript delineates our discoveries after an expedition to the Bulgarian Antarctic Base “St. Kliment Ohridski” situated on Livingston Island, Antarctica. Amplicon-based metagenomics targeting the 16S rRNA genes and ITS2 region were employed to assess the metagenomes of the bacterial, fungal, and archaeal communities across diverse sites within and proximal to the research station. The predominant bacterial assemblages identified included Oxyphotobacteria, Bacteroidia, Gammaprotobacteria, and Alphaprotobacteria. A substantial proportion of cyanobacteria reads were attributed to a singular uncultured taxon within the family Leptolyngbyaceae. The bacterial profile of a lagoon near the base exhibited indications of penguin activity, characterized by a higher abundance of Clostridia, similar to lithotelm samples from Hannah Pt. Although most fungal reads in the samples could not be identified at the species level, noteworthy genera, namely Betamyces and Tetracladium, were identified. Archaeal abundance was negligible, with prevalent groups including Woesearchaeales, Nitrosarchaeum, Candidatus Nitrosopumilus, and Marine Group II

Genotypic and phenotypic insights into virulence factors of nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria (2011-2022)

The present study aimed to explore the virulence characteristics in 221 Bulgarian nosocomial Steno-trophomonas maltophilia isolates (2011-2022) via screening for the presence of virulence genes, their mutational variability, and the corresponding enzyme activity. PCR amplification, enzymatic assays, whole-genome sequencing (WGS), and biofilm quantification on a polystyrene plate were performed. The incidence of virulence determinants was as follows: stmPr1 (encoding for the major extracellular protease StmPr1) 87.3%, stmPr2 (minor extracellular protease StmPr2) 99.1%, Smlt3773 locus (outer membrane esterase) 98.2%, plcN1 (non-hemolytic phospholipase C) 99.1%, and smf-1 (type-1 fimbriae, biofilm-related gene) 96.4%. The 1621-bp allele of stmPr1 was most frequently found (61.1%), followed by the combined allelic variant (17.6%), stmPr1-negative genotype (12.7%), and 868-bp allele (8.6%). Protease, esterase, and lecithinase activity was observed in 95%, 98.2%, and 17.2% of the isolates, respectively. The WGS-subjected isolates (n = 9) formed two groups. Five isolates possessed only the 1621-bp variant of stmPr1, higher biofilm formation ability (Optical Density at lambda = 550 nm (OD550): 1.253-1.789), as well as a low number of mutations in the protease genes and smf-1. Three other isolates had only the 868-bp variant, weaker biofilm production (OD550: 0.788-1.108), and higher number of mutations within these genes. The only weak biofilm producer (OD550 = 0.177) had no stmPr1 alleles. In conclusion, the similar PCR detection rates did not allow differentiation of the isolates. In contrast, WGS permitted stmPr1 al-leles-based differentiation. To the best of our knowledge, this is the first Bulgarian study presenting genotypic and phenotypic insights into virulence factors of S. maltophilia isolates

Comparison of Two Methods for SARS-CoV-2 Detection in Wastewater: A Case Study from Sofia, Bulgaria

Wastewater surveillance for monitoring the spread of SARS-CoV-2 remains important even in the current endemic stage of the COVID-19 outbreak. This approach has already demonstrated its value by providing early warnings of coronavirus spread in different communities. The aim of the present publication is to share relevant experience from the Center of Competence “Clean&Circle”, obtained in the development of an effective strategy for SARS-CoV-2 detection in the wastewater of Sofia, Bulgaria. Using four different RNA concentration/extraction methods, we revealed that the key hindering factor for successful viral detection was the presence of PCR inhibitors in the wastewater. The most efficient way to overcome their presence turned out to be the application of a specialized polymerase in the RT-PCR detection setup. Our data showed that using such an enzyme increases the detection efficiency from 1.9% to 70.5% in samples with a spiked control virus. We also evaluated the recovery rates of viral particles by using silica columns (71%), PEG precipitation (23%), ultrafiltration (15%), and MCE filtration (10%). These results support the international effort to unify and standardize the various techniques used for SARS-CoV-2 monitoring in wastewater