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The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n = 221) during the period 2011–2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgM+/rmlA+/rpfF+ (56.1%), followed by spgM+/rmlA+/rpfF- (28.5%), and spgM+/rmlA-/rpfF+ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 ± 0.049 to 2.065 ± 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 ± 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 ± 0.028 vs. 1.348 ± 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing sul1 in their 3′ conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections.

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.

In this study, we used the sequence of the aac(6′)-Ii gene, which is responsible for the intrinsic low-level aminoglycoside resistance of Enterococcus faecium, to design novel species-specific primers. Two oligonucleotide pairs named EF_200 and EF_120 were designed, generating amplification products with sizes of 200 bp and 120 bp, respectively. They were successfully applied for the identification of various isolates of clinical or environmental origins in both pure cultures and complex food samples. The obtained results indicated that both primer pairs permitted the highly specific, simple, fast and inexpensive detection of E. faecium isolates.

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, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter 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 cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-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.

In this research, we report the isolation of Enterococcus durans EDD2, which showed strong inhibitory activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (A...

To the Editor,Multidrug-resistant Pseudomonas aeruginosa is one of the most clinically important opportunistic pathogens, responsible for a wide variety of hospital-acquired infections. Carbapenem ...

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.

To the editor, Stenotrophomonas maltophilia is globally recognized as an emerging multidrug-resistant (MDR) pathogen in healthcare facilities, especially in the intensive care units (ICUs) where it...

To the Editor,Acinetobacter baumannii (AB) is globally recognized as a main pathogen in the modern healthcare system, especially in the intensive care units (ICUs) where it causes a wide range of n...

To the Editor,Infections caused by multidrug-resistant (MDR) Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, are of great concern for hospitalized patients because of very lim...

Objectives A total of 226 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates was collected during 2014–2016 from inpatients (age range 5–88 years) in four Bulgarian university hospitals (H1–H4) to assess their antimicrobial susceptibility and to explore carbapenem resistance mechanisms as well as the molecular epidemiology of the isolates. Methods Antimicrobial susceptibility testing, multiplex PCR, DNA sequencing and electrotransformation experiments were performed. Epidemiological typing by random amplification of polymorphic DNA (RAPD)–PCR was also performed. Results The resistance rates were as follows: imipenem, 90.7%; meropenem, 98.2%; doripenem, 100%; amikacin, 92.9%; gentamicin, 87.2%; tobramycin, 55.8%; levofloxacin, 98.2%; trimethoprim/sulfamethoxazole, 86.3%; tigecycline, 22.1%; colistin, 0%; and ampicillin/sulbactam, 41.6%. Intrinsic blaOXA-51-like genes were found in all of the isolates. The majority of the A. baumannii isolates harboured either blaOXA-23-like associated with the upstream-located ISAba1 (26.1%) or blaOXA-40/24-like (46.7%), 45 isolates (19.9%) harboured both genes, and 1 isolate harboured blaOXA-58-like surrounded by ISAba3C upstream and ISAba3 downstream. The blaOXA-58 gene was transferable by electroporation indicating its plasmid location. Epidemiological typing revealed the dissemination of nosocomial CRAB with high clonal relatedness (70% similarity threshold) belonging to six, four, three and two clusters in H1, H2, H3, and H4 hospitals, respectively. Conclusions The A. baumannii isolates studied were problematic nosocomial pathogens. Their multidrug resistance greatly limits therapeutic options. The persistence of endemic clones comprised of OXA carbapenemase-producing multidrug-resistant A. baumannii in the monitored hospitals over a period of ca. 3 years is of concern and requires continuous detailed investigations in the future.

Chronic lung infection is one of the hallmarks in cystic fibrosis (CF). Respiratory cultures from CF patients usually indicate colonization most commonly with Staphylococcus aureus, followed by Pseudomonas aeruginosa and Burkholderia cepacia, all of them listed as a classic pathogen for CF. We present a case of 15-year-old patient with CF, diagnosed at the age of 2 years due to failure to thrive and recurrent lung infections. He survived severe peritonitis at the age of 5 years, when he was colonized with P.aeruginosa for the first time. Following strict intravenous (piperacillin/tazobactam or ceftazidime) and inhaled antibiotic regimens (tobramycin, colistin and azytromycin), he is P.aeruginosa free for the last 6 years. However, in his sputum samples grew constanly Achrombacter xylosoxidans. Rigourous inhaled colistin and sulfomethaxasole/trimethoprim therapy was followed until the germ was resistant to every antibiotic tested but meropenem. Last year he was on inhaled meropenem with favorable effect – no A.xylosoxidans in his sputum from more than 6 months. His lung health was relatively stable at FVE1 of about 45-50% for almost 2 years, but this summer he had experienced new exacerbation and FEV1 drop to 26%. At that moment we found Myroides odoratus in his sputum sampled. The therapy was modified. Three months later in the sputum of the patient we found only S.aureus and some fungal colonies, his FEV1 is back at 50% and he gained 2 kilos. Myroides remains an uncommon pathogen that is ubiquitous in the environment. Clinicians should remain alert to the possibility of this one or other new emerging pathogens in CF when there is a lack of response from routine treatment

Nowadays, due to their potential application as probiotics for humans or animals, many beneficial lactic acid bacteria have been isolated from different natural environments. These include members of the genus Enterococcus - quite specific due to their ambiguous nature, varying from pathogens to probiotics. In our work we present a whole-genome sequencing (WGS)-based approach for assessing the potential of bacteriocin-producing Enterococcus isolates from beehives to serve as natural preserving agents against bacterial infections associated with honeybees. Potential Enterococcus spp. isolates from pollen granules were tested with the well diffusion assay for bacteriocin activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (AFB). Two of them gave positive results and were determined at species level by 16S rRNA genes sequencing. They were then subjected to WGS using the Illumina HiSeq platform. The resulting raw data reads were processed and further analyzed by using only freely available web-based tools (the Shovill pipeline, QUAST, BAGEL4, ResFinder, VirulenceFinder and PlasmidFinder). The analysis revealed that both of them represent clonally identical isolates of the same strain. This specific strain was named Enterococcus faecium EFD, and was genotyped by the MLST-2.0 Server. Five bacteriocin genes were found in the assembled genome, providing a possible explanation for the antimicrobial properties of the isolate. The protein nature of the inhibitory agent/s was confirmed by treatment with proteinase K. No resistance determinants for clinically important antibiotics and functional virulence factor genes were detected. The bioinformatic analyses of the draft genome sequence suggest that E. faecium EFD is not pathogenic.The observation that E. faecium EFD was present within more than one of the beehives in the apiary proposes the idea that E. faecium EFD is there as a part of the normal beehive microbiota. This finding, in combination with its antibacterial activity against P. larvae, highlights this novel isolate as a potential natural preserving agent against AFB. Furthermore, the WGS-based approach reported here proved to be very cost- and time- efficient, for screening the applicability of new pro- and prebiotic Enterococcus strains as beehive protection agents.

In this study, Enterococcus faecalis H1041 which was isolated from the traditional Bulgarian green cheese was characterized in depth by a whole-genome sequencing performed on the Illumina HiSeq platform using 2 x 150 bases paired-end reads mode. The bioinformatic analysis performed only with free online tools revealed that the strain resembles other cheese-born E. faecalis strains by its antibiotics’ resistances and virulence genotype, thus its presence within the green cheese should not be considered as a health risk. Only two antibiotic resistance genetic determinants were found, but they both carry mutations, and probably are not functional. Furthermore, 18 virulence factor genetic determinants were found, but only 6 of them possessed 100% identities with the canonical sequences, and being more or less intrinsic for the Enterococcus genus. None of them were found on contigs that contain sequences with known plasmid origin of replication. Enterolysin A gene was also detected which most probably plays role in ripening process of the Bulgarian green cheese.

Stenotrophomonas maltophilia is an increasingly prevalent opportunistic pathogen responsible for a wide range of nosocomial infections in intensive care unit patients, life-threatening diseases in immunocompromised haematology-oncology patients and chronic pulmonary infections in individuals with cystic fibrosis. Therapy of these infections is problematic due to the remarkable intrinsic antimicrobial resistance of the species and to acquired resistance to multiple antimicrobial agents. As this organism is a low-grade pathogen, the pathogenesis of S. maltophilia infections involves numerous virulence factors as well as the ability of bacterial cells to form biofilms on abiotic surfaces and host tissues. The present review summarizes the literature data regarding extracellular and cell-associated virulence factors of S. maltophilia (some of which have still not been studied in detail) and considers the basic characteristics of biofilm formation. Many virulence features such as extracellular enzymes, bacterial motility and biofilm formation are finely controlled by quorum sensing (QS) that enable the bacteria to express these virulence factors in a coordinated, cell-density-dependent manner and overwhelm the host defence mechanisms. Manipulating the QS regulatory system is a promising approach for development of new strategies for control of S. maltophilia infections.

Sir,Enterococci are generally low-virulent, but can cause serious infections in immunocompromised patients. Clonal spread of vancomycin-susceptible Enterococcus faecium at an adult haematology ward...

To the editor,Stenotrophomonas maltophilia is an environmental, Gram-negative bacterium that has been recognized as an emerging multidrug-resistant nosocomial pathogen. It is responsible for divers...

The aim of the study is to examine different thresholds of similarity coefficients for reliable clustering of clinical and dairy enterococci originating from various geographical regions in Bulgaria, based on randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) unweighted pair group method with arithmetic mean (UPGMA) analysis. For creating subsets of different strains on a relatively independent basis, was created a selection based on the presence of some bacteriocin genes. A collection of 110 Enterococcus faecalis and 51 Enterococcus faecium nosocomial strains from in-patients in five hospitals, covering the major cities of Bulgaria, and additional 11 Enterococcus faecalis and 15 Enterococcus faecium strains isolated from home-made and artisanal cheeses in different mountain regions of the country was screened for the presence of five commonly found bacteriocin genetic determinants – enterolysin A (EntlA) and bacteriocin AS-48 for E. faecalis and enterocin A (EntA), enterocin B (EntB) and enterocin P (EntP) for E. faecium. Strains carrying the same bacteriocin gene were subjected separately to RAPD-PCR, followed by UPGMA analysis. The obtained results showed that clustering should be performed with similarity coefficients above 95% in order to separate the starins based on cheeses’ or clinical origin and according to their geographical origins. Our findings indicate that the largely accepted similarity coefficient of 70% for clonal relatedness should be reconsidered and the threshold raised up.

A total of 424 Enterococcus spp. isolates from clinical samples (n = 398) and traditional Bulgarian artisanal cheeses (n = 26) were studied in order to evaluate the incidence and expression of bacteriocin genetic determinants. Structural genes coding for enterocin A (entA), enterocin P (entP), enterocin B (entB), enterocin AS-48 (as-48) and enterolysin A (entlA) were detected by polymerase chain reaction (PCR), while reverse transcription PCR was performed to establish the gene expression. Of the total isolates, 163 (38.4 %) were potentially bacteriocinogenic. The genes typical of Enterococcus faecium (entA, entB and entP) were found in 84.0, 8.4 and 15.1 % of the E. faecium isolates, respectively. Focusing on E. faecalis isolates, the frequencies of entlA and as-48 were 19.3 and 0.7 %, respectively. No significant differences in the gene presence between clinical and dairy isolates were ascertained. Bacteriocin genes were found to be expressed at transcriptional level in 83.4 % of the bacteriocinogenic Enterococcus spp. isolates: a single gene in 113 samples (69.3 %), two genes in 21 (12.9 %) and three genes in 2 isolates (1.2 %). The present investigation may serve as a basis for further studies for elucidating the spread of the bacteriocin genetic determinants in the two enterococcal populations (clinical and dairy).

Background/aim: This study aimed to investigate the correlation between the prevalence of problematic pathogens and the clinical status of women with bacterial vaginosis (BV). Materials and methods: Gardnerella vaginalis, Atopobium vaginae, and Mobiluncus spp. were detected using a multiplex PCR assay, and their role in the infection of Bulgarian women with clinically heterogeneous BV was evaluated. Results: The predominant BV-associated pathogen identified was G. vaginalis with an incidence of 98.39%, followed by A. vaginae (68.05%) and Mobiluncus spp. at 17.01%. The coexistence of A. vaginae and G. vaginalis was more common in women with discharge (in 72.04%) and in patients with chronic recurrent BV than among asymptomatic or newly diagnosed BV cases (P < 0.05). Mobiluncus spp. was detected mostly in coinfections, in association with Trichomonas vaginalis. The coinfections were predominantly related to recurrent BV and with complications (P < 0.05). Conclusion: This is the first study about the correlation between problematic pathogens and clinically heterogeneous BV in Bulgarian women. High frequency of infection with key BV-related pathogens was observed in childbearing women. The incidence was shown to often correlate with coexistent T. vaginalis, with severity of infection, and with complicated and recurrent BV after unsuccessful treatments. Screening should be considered in reproductive health programs.

Background: Across the globe, upper respiratory tract infections (URTIs) are the most prevalent cause of morbidity in childhood. Aims: The aim of our study is to analyze the incidence and etiology of bacterial URTIs in Bulgarian children, as well as the increasing antimicrobial resistance to the most common etiologic agents over a period of 17 years. Study Design: Retrospective study. Methods: The study material comprised the data from 4768 patients (aged 1-16 years) with URTI during the period from 1998-2014. Specific microbiology agent detection was performed by culture examination. Susceptibilities to the investigated pathogens were determined by the disk diffusion method and minimal inhibitory concentration according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). Polymerase chain reaction was used to detect the presence of β-lactam resistance genes. Results: We identified the following as the most common URTI bacterial pathogens: Streptococcus pneumoniae (40.94%), Streptococcus pyogenes (34.16%), Haemophilus influenzae (44.23%), Moraxella catarrhalis (39.19%) and Staphylococcus aureus (23.88%). In more than 70% of cases, a polymicrobial etiology was found. The most commonly affected individuals were pre-school-aged children, which accounted for more than 36% of all patients. During the study period, a dramatic increase in resistance to antibiotic agents was observed. The most frequent types of resistance were the enzymatic inactivation of penicillins and cephalosporins (close to 100% in staphylococci and moraxellae) and inducible macrolide-lincozamide resistance (about 20% of Gram-positive cocci). Conclusion: Due to mandatory immunization against pneumococci and H. influenzae in Bulgaria and the vast expanding resistance to the most popular antimicrobial agents changes in the etiology of URTI have recently been noted. Regular analysis of this etiological dynamic and the antimicrobial resistance of respiratory pathogens is important for choosing the correct therapy and successful treatment.

Background: Neuraminidase production in isolates of Pseudomonas aeruginosa from CF patients was described and suggested to contribute to pulmonary infection. Aim: To evaluate nan1 presence in chronically infected with P.aerugenosa CF patients and it9s impact on survival and clinical course. Material and methods: In 2008 we evaluated clinical course and state of 29 patients with cystic fibrosis (CF) aged from 7 to 17 years of age, with chronic P. aeruginosa infection. We searched for nan1 among the studied isolates of P. aeruginosa by PCR. Based on nan1 results the patients were divided in two groups – nan1 positive (nan1 (+)) and nan1 negative (nan1(-)). We reevaluated the same patients 7 years later. Results: In 2008 there was no significant difference in sex (p>0.05) or genetic mutation (p>0.05) distribution in both groups. Patients9 age also was not significantly different 14.3 ± 3.01 years in nan1(+) group (n=14) and 14.0 ± 3.1 years for nan1 (-) (n=15) (p>0.05). Period of chronic colonization was higher in nan1(+) group 5.6 ± 1.4 years vs. 3 ± 1.4 years (p=0.05) . Nan 1(+) group have more recurrent exacerbations of pulmonary disease and weaker clinical status. In 2015 all but one patient from nan1(+) group have died, while in nan1(-) group only 2 death are registered. In 2015 - four from the alive patients from nan1(-) group in 2008 have nan1(+) P.aeruginosa in their sputum, and are in worse clinical (examination and PFT) state compared to the rest 9 of this group, but similar to the only one survival from nan1(+) group. Conclusion: Chronic pseudomonas infection with strains positive for neuraminidase could be additional prognostic factor for poor prognosis.

Objectives: To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Methods: Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. Results: The overall prevalence was: esp – 44.3%, agg/asa1 – 38.4%, ace/acm – 64.3%, efaA – 85.9%, eep – 69.4%, gelE – 64.3%, hyl – 25.1%, and cylA – 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Conclusion: Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study. Keywords: Enterococcus faecalis, Enterococcus faecium, Virulence genes, Prevalence

Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that causes a variety of nosocomial infections, life-threatening diseases in immunocompromised persons and chronic pulmonary infections in cystic fibrosis patients. The organism’s virulence depends on an arsenal of cell-associated and extracellular factors determining the pathogenesis of infections as multifactorial. Most P. aeruginosa infections are both invasive and toxinogenic. Many of the extracellular virulence factors (proteases, exotoxin A, pyocyanin, siderophores, hemolysins) required for tissue invasion and dissemination of P. aeruginosa are controlled by quorum sensing (QS) that enable the bacteria to produce these factors in a coordinated, cell-density-dependent manner and overwhelm the host defense mechanisms during acute infection. Sometimes, QS also contributes to biofilm formation and thus participates in pathogenesis of chronic infection. This system is recognized to be a global regulatory network controlling the expression of a large number of virulence genes either directly or indirectly. Two-component sensor kinases such as RetS, LadS and GacS are also controlling the production of virulence factors as well as the switch from acute to chronic infection. The present review describes the known virulence determinants of P. aeruginosa, the stages of infection as well as the importance of QS in the pathogenesis of P. aeruginosa infection.

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

A total of 203 clinical isolates of Pseudomonas aeruginosa was collected during 2001–2006 from five university hospitals in Sofia, Bulgaria, to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to antipseudomonal antimicrobial agents. The antibiotic resistance rates against the following antimicrobials were: carbenicillin 93.1 %, azlocillin 91.6 %, piperacillin 86.2 %, piperacillin/tazobactam 56.8 %, ceftazidime 45.8 %, cefepime 48.9 %, cefpirome 58.2 %, aztreonam 49.8 %, imipenem 42.3 %, meropenem 45.5 %, amikacin 59.1 %, gentamicin 79.7 %, tobramycin 89.6 %, netilmicin 69.6 % and ciprofloxacin 80.3 %. A total of 101 of the studied P. aeruginosa isolates (49.8 %) were multidrug resistant. Structural genes encoding class A and class D β-lactamases showed the following frequencies: bla VEB-1 33.1 %, bla PSE-1 22.5 %, bla PER-1 0 %, bla OXA-groupI 41.3 % and bla OXA-groupII 8.8 %. IMP- and VIM-type carbapenemases were not detected. In conclusion, the studied clinical strains of P. aeruginosa were problematic nosocomial pathogens. VEB-1 extended-spectrum β-lactamases appear to have a significant presence among clinical P. aeruginosa isolates from Sofia. Carbapenem resistance was related to non-enzymic mechanisms such as a deficiency of OprD proteins and active efflux.

Introduction: In aim to achieve better infection control and possible eradication of the pathogens involved in chronic infections of patients with cystic fibrosis (CF) scientists have developed a new way to administer antimicrobials – inhalation. The first and so far the only available inhalable antimicrobial in Bulgaria is inhaled tobramycin (TOBI), introduced in 2009.We aimed to evaluate the antimicrobial susceptibility of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients before and after initiation of TOBI in the regular treatment regimen. Methodology: We have determined the minimal inhibitory concentration (MIC) of 17 antimicrobials by the E-test (LIOFILCHEM) in sputa samples of 118 CF patients for the period of 2005–2014. The results were interpreted according to the annual Clinical and Laboratory Standards Institute guidelines. Results: In the sputa of 70 patients a total of 102 P. aeruginosa isolates were found. Sixty-eight out of 102 (66.7%) were susceptible to all studied antimicrobials. We divided the isolates in two chronological groups: those collected before the introduction of TOBI as a regular treatment in 2009 and those collected after 2009. A significant reduction (p < 0,001–0,02) in susceptibility for the strains after 2009 was noted towards piperacillin (100% vs 50%), ceftazidime (100% / 77.3%), cefepime (97.9% / 68.2%), amikacin (100% / 63.6%), gentamicin (95.7% / 40.9%), tobramycin (93.6% / 59.1%) and ciprofloxacin (93.6% / 45.5%). Conclusion: The introduction of inhaled tobramycin as a regular therapy for CF patients in Bulgaria lead to a significant change in antimicrobial susceptibility of CF P. aeruginosa.

Aim: The purpose of this work is to evaluate the clinical significance of bronchoalveolar lavage (BAL) in children with asthma (A), bronchiectasis (BE) and cystic fibrosis (CF) Methods: For period of 2006-2009 we performed bronchoscopy with obtaining broncho-alveolar lavage of 60 children aged 4 to 17 years, divided in three groups: Group I - BE(10 children); Group II – A (20children); Group III – CF (30 children). All children underwent a fibrobronchoscopy with BALaspiration. Microbilogical, cytological and immunological examinations of the BAL liquid were performed. Results: Eosinophils were typical for group II. Neutrophil-dominated endobronchial inflammation was characteristic for CF and BE groups. For CF the levels of polymorphonuclear cells were 30-79%. BE children with catarrhal endobronchitis had high level of SIgA and IgG; CF children with purulent endobonchitis, had IgG levels even higher with demonstrative low levels SIgA; for children with A we found low level of SIgA and high level of IgE. As a contrast of the other groups all CF patients had significantly low levels of a 1 -antitripsin. Most common microbiology finding in CF patients was P.aeruginosa , followed by S.aureus . The patients with A didn9t have any clinical relevant pathogen, while for BE a diverse microbial flora, without any prevalent pathogen was found. Conclusions: If used properly, BAL can be a useful clinical tool that can assist in the diagnosis and follow up in children with A, BE and CF, for it reflects the extent of the chronic inflammation and chronic infection in the lungs.

Aim: To evaluate antimicrobial susceptibility of Pseudomonas aeruginosa , isolated from CF patients before and after initiation of regular therapy with inhaled tobramycin. Methods: For the period of 2006–2013 we evaluated sputa from 118 CF patients (50 females and 68 males) aged 5 to 34 years of age. We evaluated the antimicrobial susceptibility towards 17 antimicrobials, according CLSI recommendations. Results: In the sputa of 70 patients(32 males and 38 females) a total of 102 isolates of P . aeruginosa were found. Age distribution was as follows: 42,1% of all children under 9 years of age, 57,9% for children aged 10 to 16 years and 86,9% for patients over 16 years of age. 68 out of 102 (66.7%) were susceptible to all 17 studied antimicrobials. Low resistance levels were found towards: imipenem (5.9%), meropenem (3.9%), amikacin (3.9%), gentamicin (7.8%), tobramycin (5.9%), netilmicin (13.7%) and ciprofloxacin (13.7%). In Bulgaria the first inhaled tobramycin regimens were introduced in 2007 and this treatment became regular in 2009. We divided the isolates before 2009 (64) and after 2009 (38). Significant reduction (p

In patients with cystic fibrosis (CF) lung damage secondary to chronic infection is the main cause of death. Treatment of lung disease to reduce the impact of infection, inflammation and subsequent lung injury is therefore of major importance. As Pseudomonas aeruginosa is the dominant pathogen in CF patients it has been the major target of all treatment strategies, possible antibiotic regimens and recommendations for years. More sophisticated antibiotic therapies introduced over the last decades have helped to improve the prognosis in cystic fibrosis, but then new multidrug-resistant pathogens emerged. We present a case of cystic fibrosis in a 16-year-old boy with pulmonary exacerbation due to colistin-resistant Stenotrophomonas maltophilia. This case raises some interesting questions regarding the antibiotic policy and treatment options in our country for patients with CF and multidrug-resistant strains. Colistin is used at present in Bulgaria as a strategic last option for the CF patients but with the advent of new more drug-resistant strains therapeutic approach should change - for instance, there should be restrictions imposed on the use of levofloxacin and trimethoprim/sulfamethoxazole which are regarded as “cheap and not so potent” antibiotics suitable for any infection and use them only in strict dependence on the respective culture results.