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Moraxella catarrhalis has been recognized as an important cause of upper respiratory tract and middle ear infections in children, as well as chronic obstructive pulmonary disease and chronic bronchitis in adults. We aim to study the clonal structure, antimicrobial resistance, and serotypes of M. catarrhalis strains recovered from patients of different ages. Nasopharyngeal swabs, middle ear fluid, and sputum samples were collected. In vitro susceptibility testing was performed according to EUCAST criteria. The monoclonal Ab hybridoma technique was used for serotyping. All strains were subjected to MLST. The studied population demonstrated susceptibility to all tested antimicrobials M. catarrhalis strains, with the majority being serotype A (90.4%), followed by B (6.8%), and C (2.7%). We observed a predominant clonal complex CC224 (21.9%) along with other clusters including CC141 (8.2%), CC184 (8.2%), CC449 (6.8%), CC390 (5.5%), and CC67 (2.7%). Two primary founders, namely, ST224 and ST141, were identified. The analyzed genetic lineages displayed diversity but revealed the predominance of two main clusters, CC224 and CC141, encompassing multidrug-resistant sequence types distributed in other regions. These data underscore the need for ongoing epidemiological monitoring of successfully circulating clones and the implementation of adequate antibiotic policies to limit or delay the spread of multidrug-resistant strains in our region.

Haemophilus influenzae is one of the main bacteria responsible for otitis media (OM) among children worldwide. We aimed to estimate the distribution of encapsulated and non-capsulated variants (NTHi), biotypes, antibiotic susceptibility, and molecular epidemiology of H. influenzae isolates recovered from pediatric OM cases in Bulgaria.Capsule detection was done by PCR for bexB gene, absent in NTHi. All encapsulated strains were subjected to PCR serotyping. MIC susceptibility testing was performed according to the criteria of EUCAST. MLST was conducted for all 71 OM isolates.The capsule detection and PCR - serotyping disclosed a predominance of NTHi (90.1%) and a few "a", "f", and "c" types. Biotype I was the most widespread (42.3%). β-lactam resistance was found in 35.2% of the isolates. MLST represented heterogenic population structure, whereas the most represented clonal complexes belonged to ST-3, ST-57, ST-105, and ST-1426. 42.3% of the STs showed relatedness to globally represented clones, and 11.3% displayed affiliation to international type 2.Most of the H. influenzae isolates recovered from children with otitis media were non-typable strains from biotype I. The examined population structure was genetically diverse, with a predominance of international type 2 isolates.

The study aimed to evaluate the presence of pili in non-invasive pediatric pneumococcal isolates and to elucidate possible links with genetic lineages, serotypes, and antimicrobial resistance. We examined 147 Streptococcus pneumoniae isolates from children with respiratory tract infections and acute otitis media. Serotyping was performed by latex agglutination and capsule swelling reaction. Serogroup 6 was subjected to PCR-serotyping. Minimum inhibitory concentrations were determined according to EUCAST breakpoints. PCRs for rlrA and pitB genes were performed to detect a presence of type 1 and type 2 pili. MLST was conducted to define the clonal structure of the piliated strains. Almost all children (96.5%) were vaccinated with the pneumococcal conjugate vaccine PCV10. We detected 76.8% non-PCV10 - serotypes (NVTs) and 14.3% PCV10 serotypes. The predominant serotypes were NVTs: 19A (14.3%), 6C (12.2%), 3 (9.5%), 15A (7.5%) and 6A (6.8%). PI-1 was detected among 10.9% non-PCV10 serotypes 6A, 6C, and 19A and 6.1% PCV10 serotypes 19F and 23F. Type 2 pili were not found in the studied population. High levels of antimicrobial nonsusceptibility to erythromycin (58.5%), oral penicillin (55.8%), clindamycin (46.9%), trimethoprim-sulfamethoxazole (45.6%), tetracycline (39.5%) and ceftriaxone (16.3%) were revealed. The multidrug-resistant strains (MDR) were 55.1%. MLST represented 18 STs and three CCs among the piliated pneumococci: CC386, CC320, and CC81. More than half of the piliated strains (56.0%) belonged to successfully circulating international clones. PI-1 was associated mainly with MDR 6A, 6C, 19A, 19F, and 23F isolates from the widespread CC386, CC320, and CC81.

Background: Invasive infections caused by methicillin resistant Staphylococcus aureus and coagulase-negative staphylococci (MRSA/MRSCoN) require fast, adequate treatment., The Aim: of this study was to develop a faster protocol for direct detection of MRSA/MRSCoN in blood cultures and in abscess punctures based on mecA and species specific identification of S. aureus by polymerase-chain reaction (PCR)., Materials and Methods: We examined 77 growth-positive BACTEC blood cultures and 50 abscess punctures by routine microbiological assay and simultaneous PCR detection of MRSA/MRSCoN. The specificity of the PCR was evaluated by using DNA from another 15 microbial species for negative controls. We determined the minimum inhibitory concentration (MIC) of oxacillin, vancomycin, tigecycline, linezolid, levofloxacin, clindamycin, and erythromycin against the S. aureus isolates using the E-test., Results: In the blood cultures, the two methods detected 39.3% of MRSA, and 93.9% of MRCoNS. In the punctures, the PCR assay identified 20.9% of MRSA and 79.2% of MSSA. In the puncture cases, there were three PCR MRSA positive and culture negative samples. Screening for susceptibility to 14 antimicrobial agents demonstrated significantly higher (p<0.05) methicillin resistance in blood culture isolates than in the puncture ones (39.3% and 20.0%, respectively)., Conclusion: The new PCR protocol was very fast and specific. It was more sensitive in detecting MRSA from abscess punctures than the routine microbiological techniques. This protocol will speed up the right choice of empirical therapy, which is extremely important for saving patients’ lives., (This is an open access article distributed under the terms of the CC0 Public Domain Dedication.)

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.

Streptococcus pyogenes, or group A streptococcus (GAS), is the main etiological agent of bacterial tonsillopharyngitis and a common cause of a wide variety of other mild to severe infections., Objectives: Objectives of the present study was to determine and evaluate the distribution of genetic mechanisms associated with certain phenotypes of macrolide resistance in Bulgarian GAS isolated during the years of 2013-2016., Methods: All GAS strains were screened for the macrolide resistance genes erm(A), erm(B) and mef(A), using multiplex polymerase chain reaction (PCR). The minimal inhibitory concentrations (MICs) of erythromycin, azithromycin, clarithromycin, clindamycin were determined by E-tests., Results: Almost 23% of GAS isolates obtained in 2013-2014 and near 40% of them in 2015-2016 contained various elements of resistance. The predominant gene was mef(A), which encodes an efflux pump (M-phenotype), identified in 57.84% of the macrolide-resistant strains. The next frequently prevalent mechanism was a combination of mef(A) and erm(B) in 22.55%, which determined high-level inducible or constitutive resistance to macrolides, lincosamides and streptogramins (iMLSB or cMLSB). The highest MIC value (>256mg/L) was detected in association with erm(B) (p<0.05). The MIC range was observed to be much higher in the isolates with combinations of resistance genes vs. those with mef genes alone (p<0.05)., Conclusion: The data about the distribution and prevalence of macrolide resistance mechanisms obtained in this study can help in the treatment of persistent and recurrent GAS infections and in the correct choice of empiric therapy., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: No conflict of interest was declared by the authors.

Aim: The aim of the present study was to evaluate the bactericidal effects of different disinfection methods against microbial biofilms grown in root canals., Methods: A total of 300 freshly-extracted human teeth were infected with microbial pathogens. The biofilm formation and the effects of laser therapy, photodynamic therapy (PDT), iontophoresis, and disinfection with irrigating solutions were evaluated by counting the generations of microbial cells in the samples of root canals and by scanning electron microscopy., Results: Enterococcus faecalis and other Gram-positive cocci demonstrated highest sensitivity to the methods of antibacterial action compared here. In most of the cases observed, the antibacterial treatment was less effective against Gram-negative bacteria in dental biofilms. The biofilms that were most difficult to eliminate were those formed by Pseudomonas aeruginosa. Iontophoresis treatment with iodine and chemical disinfection with hypochlorite and chlorhexidine demonstrated the most powerful bactericidal effect. When PDT was applied with Fotosan as a photosensitizer, better disinfection was achieved in comparison to the other lasers alone., Conclusion: The comparison of these different strategies for endodontic treatment showed that hypochlorite, followed by the chlorhexidine irrigant in our experimental conditions, gave the most satisfactory results against the formation of bacterial biofilms in the root canals., (© 2015 Wiley Publishing Asia Pty Ltd.)

Background: Bacterial vaginosis (BV) is the most common cause of vaginal discharge in women of reproductive age. The purpose of this study was to determine the frequency of BV in Bulgarian pregnant and nonpregnant women from several age ranges and to compare three different laboratory methods for Gardnerella vaginalis detection in patents suffering from BV., Methods: Between September 2011 and June 2012, 809 women of 16-40 years of age separated in two major groups: nonpregnant - 469 (355 with and 114 without symptoms) and pregnant - 340 (213 and 127 respectively) were enrolled for the study. The women underwent three different laboratory tests simultaneously: scoring of Gram staining of vaginal smear, culture, and polymerase chain reaction (PCR) assay for G. vaginalis., Results: The microscopic method detected high frequency of BV in symptomatic (57%) whereas only a minority of asymptomatic subjects (14%) were detected. G. vaginalis-associated BV was diagnosed in approximately equal proportions when evaluated with PCR and microscopic method for both pregnant and nonpregnant women. The comparative analysis of microscopic evaluation, culture and PCR assays demonstrated greater concurrence (about 90%) between Gram staining and PCR detection for BV, than both methods compared to culture. The combination of microscopy and PCR turned out to be very reliable and repeatable for detecting G. vaginalis-associated BV., Conclusions: This is the first comparative investigation on the epidemiology of G. vaginalis-associated BV in Bulgaria. The established highest frequency in the young Bulgarian women (21-30 years) is alarming and should be considered in prophylaxis and reproductive programmes., (Copyright © 2013 Elsevier Editora Ltda. All rights reserved.)

Background and Aims: A total of 115 Moraxella catarrhalis isolates from patients with lower respiratory tract and otorhinolaryngeal infections, as well as healthy carriers, were collected to study the prevalence of outer membrane virulence and resistance encoding genes and lipooligosaccharide (LOS) serotypes., Methods: PCR technique was used to determine the presence of genes ompB2 (encoding uptake of vital iron), ompCD and ompE (for adhesion, complement resistance and transporter proteins), bro1 and bro2 (for β-lactamases). Serotyping was carried out by monoclonal antibodies (MAb) against LOS serotypes A, B and C., Results: The frequency of genes determining virulence factors were ompE 82.61%, ompCD 70.43%, ompB2 43.48%, and bro 98.26%. Dissemination of virulence genes varied according to the type of infections and carrier state., Conclusions: The present study revealed the presence of a greater number of virulence factors in isolates from patients compared to that in strains from healthy individuals. In the strains of patients with bronchopulmonary infections, a combination of ompB2-ompCD-ompE genes was predominant. In patients suffering from otorhinolaryngeal infections, isolates with combination ompCD-ompE prevailed. Isolates from the control group revealed single ompCD or ompE genes or none of the virulence genes tested. The greatest proportion (91.30%) of β-lactamases was encoded from bro1 genes. Serotype A was the most frequent (70.43%) serotype. No correlation was found between the strain serotype and presence of the virulence and β-lactamase encoding genes tested., (Copyright © 2010 IMSS. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.

New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b). The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium. When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains.

Complexes of copper(II), nickel(II) and iron(III) with beta-lactamase inhibitor sulbactam have been synthesized, characterized and identified by elemental analysis, IR and 1H NMR spectroscopy. These complexes have been then tested for their in vitro antibacterial activity in combination with ampicillin against various bacterial species.