Phenotypic and genotypic characteristics of non-invasive S. pneumoniae isolates recovered from PCV10-vaccinated children in Bulgaria.

PURPOSE: The non-invasive pneumococcal disease (NIPD) is a common infection during childhood. We aimed to define the clonal spread of pediatric non-invasive isolates recovered during the PCV10-period in Bulgaria concerning the serotype and antimicrobial susceptibility. MATERIALS AND METHODS: Serogrouping/serotyping were performed using latex agglutination and capsular swelling reaction. Serogroup 6 strains were subjected to serotype-specific PCR's. The antibiotic susceptibilities were assessed by broth microdilution. MLST was performed to define the clonal composition. RESULTS: We analyzed 154 pediatrics non-invasive S. pneumoniae isolates. The PCV10-vaccinated children were 94.1%. We disclosed 88% non-vaccine serotypes (NVTs) and 12% PCV10 - serotypes. All common serotypes among PCV10-vaccinated children (n â€‹= â€‹145) were non-vaccine types (NVTs): 19A (13.8%), 6C (11.7%), 3 (9.6%), 15A (8.3%) and 23A (5.5%). Antimicrobial non-susceptibility showed highest levels in erythromycin (50.0%), oral penicillin (49.4%), clindamycin (45.4%), trimethoprim-sulfamethoxazole (43.5%), tetracycline (42.2%), and ceftriaxone (14.3%). The multidrug-resistant strains (MDR) were 51.3%. MDR-serotypes were 6C (20.2%), 19A (17.7%), 15A (11.4%), 19F (10.1%), and 23A (8.9%). MLST presented 17 clonal complexes (CCs) with prevalence of CC320, CC386, CC505, CC8029 and CC2613 clustered 83% MDR isolates. CONCLUSIONS: All emergent pediatric non-invasive serotypes in our geographic area during the studied PCV10-period were NVTs (19A, 6C, 3, 15A, and 23A). The fifth widespread CCs: CC320, CC386, CC505, CC8029 and CC2613 clustered 83% MDR isolates. Future surveillance of vaccine-induced changes in the clonality and the antimicrobial resistance of the pneumococcal population is needed.

Rise of multidrug-resistant Streptococcus pneumoniae clones expressing non-vaccine serotypes among children following introduction of the 10-valent pneumococcal conjugate vaccine in Bulgaria.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) have reduced the incidence of pneumococcal disease, but non-vaccine serotypes are of concern, particularly if antimicrobial-resistant. This study retrospectively evaluated the serotype-specific clonality of paediatric multidrug-resistant (MDR) invasive and non-invasive Streptococcus pneumoniae isolates collected following PCV10 introduction (2011-2017) in Bulgaria. METHODS: Capsular types, drug resistance patterns and multilocus sequence typing (MLST) of the most common MDR S. pneumoniae serotypes sampled from children were determined. RESULTS: Overall, the rate of MDR pneumococci was 44.6% (107/240). The most common serotypes among MDR strains were 19F (25.2%), 19A (19.6%), 6C (13.1%), 6A and 23A (6.5% each) and 15A (4.7%), contributing 75.7% of all MDR strains. With the exception of serotype 19F, the remaining serotypes were non-PCV10 types. Among MDR pneumococci, the most frequently found sequence types were ST320 (30.4%; 19A and 19F), ST386 (12.7%; 6C and 6A) and ST8029 (5.1%; 23A). The majority of MDR STs (74.7%) belonged to PMEN clonal complexes, of which the most common were CC320 (Taiwan19F-14), CC315 (Poland6B-20) and CC180 (Netherlands3-31), accounting for 43.0%, 13.9% and 5.1%, respectively. In the post-vaccine period, a shift in the genetic structure of serotype 19A was found, with a significant increase of PMEN-14 (CC320) and a concurrent decrease of the major clone Denmark14-32 (CC230) observed prior to PCV10 introduction in Bulgaria. CONCLUSIONS: Clonality was found behind the wide distribution of MDR capsular types 19A, 6C, 23A and 3 following vaccine introduction, and a highly multiresistant and virulent clone Taiwan19F-14/ST320 has emerged as a common pathogen in children.

Bacteriocin-like inhibitory activities of seven Lactobacillus delbrueckii subsp. bulgaricus strains against antibiotic susceptible and resistant Helicobacter pylori strains.

The aim of the study was to detect anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains by four cell-free supernatant (CFS) types. Activity of non-neutralized and non-heat-treated (CFSs1), non-neutralized and heat-treated (CFSs2), pH neutralized, catalase-treated and non-heat-treated (CFSs3), or neutralized, catalase- and heat-treated (CFSs4) CFSs against 18 H. pylori strains (11 of which with antibiotic resistance) was evaluated. All GLB strains produced bacteriocin-like inhibitory substances (BLISs), the neutralized CFSs of two GLB strains inhibited >81% of test strains and those of four GLB strains were active against >71% of antibiotic resistant strains. Two H. pylori strains were BLIS resistant. The heating did not reduce the CFS activity. Briefly, all GLB strains evaluated produced heat-stable BLISs, although GLB and H. pylori strain susceptibility patterns exhibited differences. Bacteriocin-like inhibitory substance activity can be an advantage for the probiotic choice for H. pylori infection control. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains was evaluated by four cell-free supernatant (CFS) types. The GLB strains produced heat-stable bacteriocin-like inhibitory substances (BLISs) with a strong anti-H. pylori activity and some neutralized, catalase- and heat-treated CFSs inhibited >83% of the test strains. Bacteriocin-like inhibitory substance production of GLB strains can render them valuable probiotics in the control of H. pylori infection.

Study on the Composition of Biogenic Iron-Containing Materials Obtained Under Cultivation of the Leptothrix sp. on Different Media.

The biogenic iron oxide/hydroxide materials possess useful combination of physicochemical properties and are considered for application in various areas. Their production does not require special investments because these compounds are formed during cultivation of neurophilic iron bacteria. Bacteria from genus Leptothrix develop intensively in the Sphaerotilus-Leptothrix group of bacteria isolation medium and feeding media of Fedorov and Lieske. These media are different in their composition which determined the present study as an attempt to clear up the reasons that define the differences in the composition of the laboratory-obtained biomasses and the natural biomass finds. FTIRS, Mössbauer spectroscopy, and XRD were used in the research. Comparative analysis showed that the biomass and control samples contain iron compounds (α-FeOOH, γ-FeOOH, ß-FeOOH, γ-Fe2O3) in different ratios. The biomass samples were enriched in oxyhydroxides of higher dispersion. Organic residuals of bacterial origin, SO4, CO3, and PO4 groups were registered in the biogenic materials.

[Risk factors for bacterial vaginosis - a 3-year study in four medical centers].

BACKGROUND: Bacterial vaginosis (BV) is the most common infection of the lower genital tract among women of reproductive age. The purpose of this study was to determine the frequency of BV and the important etiological agent Gardnerella vaginalis in Bulgarian patients of different age groups, as well as, the risk factors forBV METHODS: One thousand five hundred and twenty-three (1523) women aged 16 to 45 years without previous antimicrobial therapy were included in this study. The methods used were: scoring of Gram staining of vaginal smear and polymerase chain reaction (PCR) assay for G. vaginalis. RESULTS: Positive for BV according to the microscopic examination criteria were 47.80% from the 1523 tested women. In 88.87% from the positive samples G. vaginalis was detected using PCR, thus demonstrating high comparability of the both methods and the leading role of G. vaginalis. The most affected with BV is the age group of 21-25 years (33.21% of all subjects with BV). The most important detected risk factors were: smoking (more than 55% with BV), single marital status (only 15% with BV are married), more than I sexual partner (more than 36% had changed the sexual partner), early onset of sexual activity (75% of B V asocciated subjects started their sexual activity by the age 15-18 years). CONCLUSIONS: The established early age for aquiring BV among Bulgarian women is very important and alarming sign. This is the first study on the etiological role of G. vaginalis and on the risk factors for BV in Bulgaria.

[Reliable microbiological diagnosis of vulvovaginal candidiasis].

INTRODUCTION: Vulvovaginal candidiasis is common infection among those affecting the vulva and vagina. Is caused by the perpesentatives from the genus Candida, in most cases C. albicans (85-90%). An increase in the percentage of the so-called non-albicans agents is seen and these pathgogens are often resistant to the most commonly used in the practice antifungals. Faulty diagnosis, incorrect use of azoles, and self-treatment lead to selection of resistant strains and recurrent infections. AIM: Identification of Candida species associated with vulvovaginal candidiasis by conventional and PCR techniques. MATERIALS AND METHODS: For six months a total number of 213 vaginal secretions were tested applying Gram stain and cultivation on ChromAgar. API Candida fermentation tests and API 20CAUX assimilation tests were performed for the identification of the bacteria. Extraction of DNA of all the smears with subsequent PCR detection of different Candida species were done. RESULTS: 80.7% materials showed presence of blastospores and/or hyphae. Positive culture results were detected in 60 (28.2%) samples. The species specific identification revealed presence of C. albicans in 51 (85%) smears, C. glabrata--in 8 (13.3%), C. krusei--in 2 (3.3%), and S. cervisie--in 1 (2.1%). The PCR technique confirmed the results of the conventional methods. It is worth to mention that 51 of the tested smears were positive for G. vaginalis using additional PCR. CONCLUSIONS: The correct diagnosis of the cause of vulvovaginal candidiasis helps in the correct choice of appropriate antifungal therapy and prevents development of recurrent infections and consequences. The PCR based method is rapid, specific and sensitive. It perfectly correlates with the results from the conventional diagnostic tests so it could be selected as a method of choice for the diagnosis of vulvovaginal candidiasis.

Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates in Bulgaria before the introduction of pneumococcal conjugate vaccine.

The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced as a mandatory vaccine in Bulgaria in April 2010. We report on the serotype distribution and the antimicrobial resistance of 222 invasive Streptococcus pneumoniae isolates collected from all age groups before the introduction of PCV10. PCV7, PCV10, and PCV13 covered 43.7, 59.9, and 78.8% of all invasive pneumococcal strains, and 64.2, 79.1, and 89.6% of isolates involving children less than 5 years of age. Penicillin resistance was found in 30.1% of the isolates responsible for meningitis and in 5.0% of isolates responsible for other invasive infections. Overall, erythromycin resistance was found in 19.4% of all invasive strains. The erm(B) was the most prevalent pneumococcal macrolide resistance genotype (63.2%) and dual mechanisms of both genes the erm(B) and mef(E) were detected in 15.8% of 19 erythromycin resistant isolates during the period 2006-2010. The prevalence and spread of serotypes 19F, 6B, and 19A during the last period may have contributed to the high predominance of erm(B) genotype in comparison of mef genotype, which was predominant in our country among erythromycin-resistant isolates before 2005. Continuing surveillance is required after the recent introduction of PCV10 in order to observe future developments of any serotype changes in the Bulgarian population, as well as surveillance of antimicrobial susceptibility of invasive S. pneumoniae isolates.

May Chlamydia trachomatis be an aetiological agent of chronic prostatic infection?

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. The objective of this study was to establish the presence/absence of C. trachomatis in 98 patients with chronic complaints about the prostate and to evaluate the role of this bacterium in the inflammation of the gland. We performed culture and microscopical examination of pre-massage/post-massage urine and expressed prostatic secretions (EPS). In all cases, culture on McCoy cells and polymerase chain reaction (PCR) of the EPS was performed. Based on laboratory findings in 53 cases (54.08%), Escherichia coli, Klebsiella, Enterobacter, Proteus, Pseudomonas and Staphylococcus were isolated and accepted as causative agents of chronic bacterial prostatitis. Forty-five patients were categorised as patients with chronic pelvic pain syndrome. The results from the PCR and the cell culture for detection of C. trachomatis were as follows - two positive probes detected at the same time by applying PCR and cultivation and 1 positive only by PCR but not by cultivation on the cell line. Based on these results, it is concluded that C. trachomatis is not so frequently detected in our patients. C. trachomatis may be accepted as one of the aetiological agents of chronic prostatitis and testing for this infection is highly recommended when presumption for chronic prostatitis is apparent.

Antimicrobial activity of tobramycin against respiratory cystic fibrosis Pseudomonas aeruginosa isolates from Bulgaria.

Tobramycin solution for inhalation (TSI) (Novartis pharmaceuticals) is indicated as chronic suppressive treatment for cystic fibrosis (CF) patients aged 6 years and older who are chronically infected by Pseudomonas aeruginosa . Inhaled administration of tobramycin assures high concentrations in the lungs of CF patients, improving the therapeutic ratio over that of parenteral tobramycin levels. Clinical and laboratory Standards institute (CLSI) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. Therefore, the Spanish meNSURA Group has defined specific values for inhaled tobramycin when testing CF P. aeruginosa isolates (susceptible: minimal inhibitory concentration (MIC) ≤ 64 µg/ml; resistant: ≥ 128 µg/ml). In this study the antimicrobial activity of tobramycin against 120 respiratory CF P. aeruginosa isolates was determined by high-range etest strips (LIOFILCHEM). Applying MENSURA breakpoints, 95% of the strains were categorized as susceptible. With CLSI breakpoints, the susceptibility rate decreased to 92.5%. The activity against non-mucoid P. aeruginosa was higher than that against mucoid isolates (MIC(50)=0.75 and MIC(90)=2 µg/ml vs. MIC(50)=1 and MIC(90)=4 µg/ml). The isolates obtained from patients untreated with TSI were more susceptible to the drug than those from patients receiving maintenance therapy with TSI (MIC(50)=0.75 and MIC(90) =1.5 µg/ml vs. MIC(50)=1.5 and MIC(90)=6 µg/ml). The isolates from patients with long-term P. aeruginosa colonization (over 5 years) revealed the highest tobramycin MICs (MIC(50)=1.00 and MIC(90)>1024 µg/ml). In conclusion, tobramycin has excellent in vitro activity against the studied CF isolates. Some factors such as isolate morphotype, pre-administration of TSI and duration of colonization influence its activity. Whenever TSI is considered for therapy, the CF P. aeruginosa strains categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be recategorized by using the MENSURA interpretive criteria.

Toluene oxidation on titanium- and iron-modified MCM-41 materials.

Iron- and titanium-modified MCM-41 materials, prepared by direct synthesis at ambient temperature or wet impregnation technique, were investigated using X-ray diffraction (XRD), transmission electron microscopy (TEM), temperature-programmed reduction (TPR), UV-vis diffuse reflectance, Mössbauer and FT-IR spectroscopies. Their catalytic behavior was studied in total oxidation of toluene. Materials with high surface area and well-ordered pore structure were obtained. The increase of the titanium content (up to 50%) in the bisubstituted, iron and titanium containing materials leads to partial structure collapse of the silica matrix. Finely dispersed anatase particles were also formed during the impregnation procedure. The catalytic activity of the bisubstituted materials was influenced by the method of their preparation, but higher catalytic stability could be achieved, compared to iron monosubstituted one. The nature of the catalytic active sites is discussed.

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report.

AIMS: To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method. METHODS AND RESULTS: LB cultures [4 x 10(8)-4 x 10(9) CFU ml(-1)) either were prepared in milk at their native pH, 3.8-5.0, or were adjusted to pH 6.4-7.7. At low and neutralized pH, LB strains inhibited the growth by 40-86.7% and 16.7-66.7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains. CONCLUSIONS: All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86.7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53.3 and 66.7% of H. pylori strains, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.

Chlamydia trachomatis infection as a problem among male partners of infertile couples.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease supposed to cause urethritis, epididymitis, prostatitis and infertility in men. The objective of this study was to assess the frequency of C. trachomatis infection in male partners of infertile couples at childbearing age. Sixty infertile couples and a control group of 40 healthy volunteers were included in the study. Urethral swabs were taken from all the male participants and cervical swabs from the female partners of the infertile couples. Culturing on McCoy cell line and PCR were the methods used for detection of the infection. C. trachomatis was found in five out of the 60 male urethral samples. Three of the female partners of these five positive males were diagnosed with C. trachomatis infection, too. We registered a woman with C. trachomatis infection whose partner's samples were negative for the bacterium. The control group showed one specimen positive for C. trachomatis. The frequency of C. trachomatis infection was 8.3% in the male partners of infertile couples at childbearing age when compared with 2.5% in the control group. It is most likely that infertility in the couples with chlamydial infection was due to the pathogen studied.

Extended-spectrum beta-lactamase-producing Serratia marcescens outbreak in a Bulgarian hospital.

We describe a nosocomial outbreak of extended-spectrum beta-lactamase (ESBL)-producing Serratia marcescens in a Bulgarian university hospital affecting nine patients on four wards. Phenotypic and genotypic (plasmid profile, randomly amplified polymorphic DNA analysis and amplified ribosomal DNA restriction analysis) analysis of the isolates indicated a single clone. The epidemic strain was resistant to oxyimino beta-lactams, aztreonam, aminoglycosides, tetracycline and chloramphenicol. It produced CTX-M-3 ESBL as demonstrated by isoelectric focusing, CTX-M PCR-RFLP and gene sequencing. The isolate was also found in the environment and from a nurse's hands, suggesting transmission by staff handling. The outbreak was controlled by patient isolation and intensified hand washing. This is the first report from Bulgaria describing a hospital outbreak caused by CTX-M-3 ESBL-producing S. marcescens.

Serum sperm antibodies after diarrhoeal diseases.

The origin of 'natural' anti-sperm antibodies found in fertile humans, virgin girls, and boys before puberty, is quite obscure. One hypothetical mechanism relates their existence to inflammatory gastrointestinal entities: as a result of the disease, cross-reactive antibodies produced against gastrointestinal flora bind spermatozoa. To test this assumption, we evaluated the level of serum sperm antibodies after diarrhoeal infections. Serum samples from 17 patients with shigellosis and 12 patients with salmonellosis were screened for anti-sperm antibodies directed against sperm surface antigens (gelatin agglutination test - GAT, tray agglutination test - TAT, sperm immobilization test - SIT), profound sperm antigens [enzyme-linked immunosorbent assay (ELISA)], and anti-bacterial antibodies (slide agglutination test - SAT) upon diagnosis (group A) and 4-35 days later (group B). The patients from group B demonstrated an increased sperm antibody incidence by GAT (20.7%), TAT (13.8%) and ELISA (31%) when compared to group A and to healthy controls, although statistically significant data were acquired only for the latter group. The absorption of positive sera with bacteria and/or spermatozoa revealed significant reactivity changes in the antibody values by GAT and TAT for shigellosis, and by TAT and ELISA for salmonellosis patients. These data demonstrate increased serum sperm antibody levels in salmonellosis and shigellosis patients.

Prevalence and macrolide resistance phenotypes and genotypes among clinical isolates of Streptococcus pneumoniae collected in Sofia, Bulgaria from 2001 to 2005.

A total of 328 clinical isolates of Streptococcus pneumoniae were analyzed to determine the rate of macrolide and penicillin resistance as well as macrolide resistance phenotypes and genotypes. Erythromycin resistance was found in 81 pneumococcal isolates (24.7%) and 10.7% of isolates were clindamycin resistant. The prevalence of penicillin G-intermediate (minimum inhibitory concentrations, MICs, 0.125 to 1 microg/ml) and penicillin-resistant (MICs, >or=2 microg/ml) S. pneumoniae isolates was 25.6% and 13.7%, respectively. The rate of ceftriaxone-intermediate and ceftriaxone-resistant strains was 2.7% and 1.2%, respectively. Among erythromycin-resistant S. pneumoniae isolates, strains harboring mef(A) genes (n=42; 51.8%) were found to be predominant over strains with erm(B) genes (n=34; 42.0%). One (1.2%) isolate carried both erm(B) and mef(A), while 4 (4.9%) isolates carried L4 protein mutations. By using the erythromycin, clindamycin and rokitamycin triple-disk test, 42 strains were assigned to the M phenotype of macrolide resistance, 31 isolates were assigned to the partially inducible (iMcLS) phenotype, 4 were assigned to the constitutive (cMLS) phenotype. Four strains with L4 gene showed a rare phenotype with the triple-disk test. Serotyping of S. pneumoniae isolates suggested that serotype (or serogroup) 14, 6 and 19 were predominant (81.5%) among erythromycin-resistant strains. Among mef(A) positive isolates serotype 14 was predominant, among erm(B) positive isolates serogroups 6 and 19 were the most prevalent.

Widespread detection of VEB-1-type extended-spectrum beta-lactamases among nosocomial ceftazidime-resistant Pseudomonas aeruginosa isolates in Sofia, Bulgaria.

A total of 132 ceftazidime-resistant clinical isolates of Pseudomonas aeruginosa were collected during 2001-2005 from 5 university hospitals in Sofia, Bulgaria to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to beta-lactams. Antimicrobial susceptibilities were detected by a disk diffusion method and E-test. Polymerase chain reaction amplification and sequencing of bla(VEB-1 )and bla(PER-1 )were performed. The antibiotic resistance rates were: to piperacillin 90.2%, piperacillin/tazobactam 52.3%, ceftazidime 94.7%, cefepime 88.6%, cefpirome 98.5%, aztreonam 85.6%, imipenem 66.6%, meropenem 63.6%, amikacin 81.1%, gentamicin 84.8%, tobramycin 89.4%, netilmicin 57.6%, ciprofloxacin 83.4%. Structural genes for VEB-1 extended-spectrum beta -lactamases (ESBLs) were found in 75 (56.8%) of the isolates. PER-1 ESBLs were not detected. The VEB-1-producing strains were more resistant than VEB-1 non-producers to amikacin, gentamicin, tobramycin and ciprofloxacin ( P<0.001). VEB-1 appears to have a significant presence among ceftazidime-resistant P. aeruginosa isolates from Sofia.

[Cervicovaginal infections during pregnancy as a risk factor for preterm delivery].

OBJECTIVE: To establish the incidence of cervicovaginal infections in patients with preterm and term deliveries. MATERIALS AND METHODS: 88 pregnancies with no previous detectable risk factors have been included in a retrospective study. 48 patients delivered at term and 40 had delivery between 24 and 37 weeks of gestation. A vaginal swab for bacterial infections and a cervical swab for detection of Chlamydia trachomatis infection, using PCR, have been performed in each case. The vaginal ecosystem has been appreciated according Nungent criteria. RESULTS AND CONCLUSIONS: C. trachomatis has been detected in 5% (2/40) of preterm deliveries and in 2.08% (1/48) of term deliveries. In preterm delivery group bacterial vaginosis has been isolated in 15% (6/40), Enterococcus spp. in12.5% (5/40) and Group B Streptococcus in 12.5% (5/40) of cases. In patients, delivered at term, bacterial vaginosis has been proved in 6.25% (3/48), Enterococcus spp in 8.33% (4/48), Group B Streptococcus in 4.17% (2/48) patients. Significant differences between both groups have been found in GBS and BV (p < 0.05). Although the difference C. trachomatis infection is not significantly higher in preterm delivery group, testing for C. tracomatis, using highly specific methods, along with vaginal swab are recommended as routine tests in pregnancy.