Natural Compounds as Inhibitors of Tyrosyl-tRNA Synthetase.

Tyrosyl-tRNA synthetases (TyrRSs) as essential enzymes for all living organisms are good candidates for therapeutic target in the prevention and therapy of microbial infection. We examined the effect of various polyphenols, alkaloids, and terpenes-secondary metabolites produced by higher plants showing many beneficial properties for the human organism, on bacterial aminoacylation reaction. The most potent inhibitors of Escherichia coli TyrRS are epigallocatechin gallate, acacetin, kaempferide, and chrysin, whereas the enzymes from Staphylococcus aureus and Pseudomonas aeruginosa are inhibited mainly by acacetin and chrysin. Most of them act as competitive inhibitors. Structure-activity relationship showed that the most potent flavonoid inhibitors contain hydroxyl group at position 5 and 7 of A ring and OCH3 group at position 4' of B ring.

[Reproducibility of Fim2 and Fim3 antigens determination in Bordetella pertussis by serotyping method]. / Ocena porównywalnosci oznaczen obecnosci antygenów Fim2 i Fim3 Bordetella pertussis metoda serotypowania.

INTRODUCTION: Serotyping is a commonly used method to characterize the presence of Fimbriae 2 and 3 in Bordetella pertussis strains for epidemiological purposes and optimal choice of strain composition of the pertussis whole-cell vaccine. Monoclonal antisera against Fim2 and Fim3 are recommended to be used for microplate serotyping instead ofpolyclonal. Reliable evaluation offimbriae expressed by B. pertussis strains influence interpretation of vaccine-driven strain evolution. METHODS: To evaluate the impact of tests conditions on the reproducibility of serotyping, results of serotyping based on a standardized protocol for microplate agglutination with monoclonal antisera performed in three different accredited laboratories were compared. For the study isolates of three vaccine strains of B. pertussis deposited within seed lot system originating from different liofilization lots were compared. RESULTS: Lack of the complete agreement on serotyping results among three labs might relates to the differences of media used, subjective reading, test conditions, and specificity of the reagents. CONCLUSIONS: Serotyping results should be interpreted with caution and the type of media and culture conditions used should be precisely recommended after validation studies. Inconsistent results should be confirmed using an alternative technique, eg. ELISA or by reference laboratory.

[Problems with identification of beta-hemolytic streptococcus resistant to bacitracin isolated from patients with pharyngitis]. / Problemy z identyfikacja paciorkowców beta-hemolizujacych opornych na bacytracyne izolowanych od chorych z zapaleniem gardla.

INTRODUCTION: The genus Streptococcus comprises a number of species characterized by a differential pathogenic potential. These bacteria can be considered as members of microbial physiological flora but they can also cause mild infections or severe, life threatening conditions. The majority of infections of streptococcal etiology are caused by beta-hemolysing species. The predominant causative agent of bacterial pharyngitis is Streptococcus pyogenes. This species usually doesn't give rise to any identification difficulties due to the introduction the well determined diagnostic schemes. Problems concerning laboratory identification can be, however, associated with other species of beta-hemolysing streptococci isolated from patients with pharyngitis. These streptococci can demonstrate features similar to those of S. pyogenes and share the group antygen A, such as some strains of Streptococcus anginosus and Streptococcus dysgalactiae subsp. equisimilis. The determination of sensitivity to bacitracin, which is a feature typical of S. pyogenes, is the basic test useful for its preliminary identification. Nevertheless, the identification of some strains by this test can give rise to incompatibility. The aim of the study was characterisation of beta-hemolysing streptococci resistant to bacitracin isolated from patients with pharyngitis. The examined bacterial strains caused identification problems by the use of routine diagnostic methods. METHODS: The material included 14 streptococcal strains resistant to bacitracin which were isolated from adult patients suffering from pharyngitis. The bacteria were cultured on media dedicated for the species. The following routine diagnostic tests were used for the bacterial identification: sensitivity to bacitracin (0.04 U/disc), CAMP test, determination of the group antigens A, B, C, D, F and G (Slidex Strepto-Kit), and determination of biochemical features by the API 20 STREP test (bioMèrieux). The sensitivity of streptococcal isolates to antibiotics (penicillin, clindamycin, erythromycin, tetracycline, vancomycin, ofloxacin) and trimethoprim/sulfamethoxazole, was determined by the disc diffusion method on the Mueller-Hinton agar with 5% sheep blood (the inoculum-0.5 McFarland). RESULTS: Among the 14 isolates resistant to bacitracin, 6 isolates of S. pyogenes, 6 isolates of S. constellatus, and 2 isolates of S. dysgalactiae subsp. equisimilis were identified. All isolates were sensitive to penicillin and vancomycin. One isolate ofS. pyogenes demonstrated constitutive MLSB resistance mechanism. Seven isolates were resistant to tetracycline: S. dysgalactiae subsp. equisimilis (3 isolates), S. constellatus (3), and S. pyogenes (1). The number of isolates resistant to trimethoprim/sulfamethoxazole was as follows: S. pyogenes (6) and S. dysgalactiae subsp. equisimilis (1), whereas four isolates were resistant to ofloxacin. CONCLUSIONS: The bacitracin test cannot be used as the only test for the laboratory identification of S. pyogenes even if it is combined with the determination of the Lancefield group antigen due to the existence of bacitracin resistant S. pyogenes strains. Therefore, it is necessary to perform biochemical commercial tests in addition to routine phenotypic tests. Isolation of beta-hemolysing streptococci other than S. pyogenes from patients with pharyngitis confirms their role in the etiology of this infection. Taken into account the significance of determination of sensitivity to bacitracin for the preliminary identification of S. pyogenes, it is necessary to standardize this test, which will make obtaining of the comparability of results possible.

[Wound infections due to opportunistic corynebacterium species]. / Zakazenia ran wywolane przez maczugowce reprezentujace gatunki bakterii uwazanych za oportunistyczne.

Wound infections are often due to endogenous bacterial flora which penetrates into a site of injury. The establishment of the etiologic agent can be problematic, especially when opportunistic bacteria are present, suggesting contamination of clinical material. Among bacteria that can cause such diagnostic problems are opportunistic Corynebacterium spp. and coryneforms colonizing skin. The aim of the study was to analyze the 24 clinical samples collected from wounds of different location, with Gram positive rods isolated in numbers suggesting the cause of infection. Bacterial identification was performed by API Coryne and additional biochemical tests (API ZYM, API NE). It was detected that the commonest species isolated were: C. amycolatum (29.2%), C. striatum (16.7%), C. group G (16.7%) and Brevibacterium spp., C. jeikeium, C. urealyticum, C. group F1. The drug susceptibility testing was performed by E-test method. Among isolated strains, 83.3% were simultaneously resistant to erythromycin and clindamycin. In 75% cases resistance to co-trimoxazole was noted, in 71.7% resistance to chloramphenicol and in 16.7% resistance to beta-lactams were detected. In presented study the high percentage of strains resistant to macrolids and linkosamids (MLSB) was noted. All strains were susceptible to vancomycin and teicoplanin.

Breakthrough in penicillin resistance? Streptococcus pneumoniae isolates with penicillin/cefotaxime MICs of 16 mg/L and their genotypic and geographical relatedness.

OBJECTIVES: To phenotypically and genotypically characterize 11 strains (isolated in four different centres) exhibiting penicillin MIC of 8-32 mg/L among isolates of the SPICE project. Nine isolates were from Romania (9/162; 5.56%) and two from Poland (2/305; 0.66%). METHODS: In vitro susceptibility was determined in triplicate by microdilution (CLSI guidelines), and additionally, MICs of penicillin, cefotaxime and amoxicillin were confirmed in triplicate by agar dilution. Multilocus sequence typing (MLST), PFGE and gene amplification and sequencing were performed. RESULTS: For the nine Romanian isolates, MICs were >/=16 mg/L for penicillin, cefotaxime and amoxicillin, >/=32 mg/L for cefuroxime and cefpodoxime, 4-8 mg/L for cefditoren and >/=128 mg/L for erythromycin and gentamicin. All isolates were non-susceptible to imipenem (MIC = 0.5-1 mg/L) and susceptible to levofloxacin (MIC = 0.5-1 mg/L) and vancomycin (MIC = 0.25-0.5 mg/L). These Romanian strains presented a new cluster in the 595-600 region of PBP2X (YSGIQL-->LSTPWF) conferring 98% homology with Streptococcus mitis PBP2X, with a new MurM allele (seven strains) with eight amino acid changes versus R6. PBP nucleotide sequences were highly conserved suggesting a common origin. Allelic profiles of two strains gave sequence type 321, three strains exhibited a single- and four a double-locus variance. MLST-predicted serotype was 23F in all but one strain (19F), but three strains were 19A by Quellung. CONCLUSIONS: The multidrug high resistance (precluding adequate oral therapy in children), its origin, the prevalence found in Romania and the presence of non-vaccine (7-valent) serotypes should worry the medical community because of a possible clonal diffusion that would limit therapeutic alternatives.

Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate.

Lipooligosaccharides (LOS) isolated from Bordetella pertussis strains 186 and 606 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonance (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS in B. pertussis is heterogeneous. The pentasaccharide was selectively cleaved from LOS of B. pertussis strain 186, purified, and covalently linked to a monomer fraction of tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete Freund's adjuvant yielded immunoglobulin G antibodies that were reactive with the LOS. These antibodies reacted strongly with B. pertussis LOS possessing the complete dodecasaccharide, as determined by an enzyme-linked immunosorbent assay, immunoblotting, and flow cytometry with intact, live bacterial cells. The binding epitope within the pentasaccharide was investigated by saturation transfer difference (STD) NMR spectroscopy. Protons H-1 and H-4 of the terminal alpha-D-GlcpNAc and proton H-6 and protons of an N-methyl group at H-4 of 3-substituted beta-L-FucpNAc4NMe exhibited the largest saturation transfers. STD NMR experiments confirmed that the immunodominant epitope recognized by the antineoglycoconjugate antibodies is located predominantly in the distal trisaccharide of B. pertussis 186 LOS. The antipentasaccharide antibodies induced by the conjugate inhibited the secretion of tumor necrosis factor alpha, interleukin-6, and NO by LOS-stimulated J774A.1 cells.

The new strategy for allele identification of the genes coding for pertussis toxin subunit S1 (ptx S1) and pertactin (prn) in Bordetella pertussis.

Bordetella pertussis strains demonstrate polymorphism in toxin subunit S1 (PT S1) and pertactin (Prn), which belong to major protective antigens of the pathogen. Changes in the distribution of particular alleles of ptxS1 and prn genes in local B. pertussis populations have been proposed as possible factors influencing the vaccination effectiveness. We have developed a new methodology for the identification of the alleles, which eliminates the necessity of DNA sequencing. The approach is based on the evaluation of the number of sequence repeats and detection of specific nucleotides at polymorphic sites of the genes, and utilizes products of their full or partial PCR amplification. The approach is available for a laboratory with standard equipment. The total conformity of the strategy with the DNA sequencing-based approach was proved on the full set of reference strains and a group of Polish clinical isolates. The new methodology was used to investigate a collection of 120 Polish B. pertussis strains isolated from the 1960s to 2001. Similarly to findings from other countries and to earlier Polish data, the tendency to change the vaccine types of PT S1 and Prn by the antigenically different ones was observed.