Persistence of non-typeable Haemophilus Influenzae in the pharynx of children with adenotonsillar hypertrophy after treatment with azithromycin.

This study was performed in children with adenotonsillar hypertrophy to evaluate the effect of azithromycin (AZT) on the presence of NTHi in monocyte/macrophages (CD14(+) cells) of adenoids/tonsils and the persistence of NTHi after adenotonsillectomy. A total of 36 pediatric patients participated in the study: 20 children were treated with AZT before adenotonsillectomy, and 16 children did not receive the antibiotic prior to surgery. NTHi were identified by culture and PCR in swabs and tissue samples. NTHi was detected in the lysates of CD14(+) cells by fluorescence in situ hybridization (FISH) and by culture. The molecular typing was used to cluster NTHi isolates from each child. The intracellular NTHi was found in 10 (62.5%) untreated patients and was identified in three (15%) azithromycin-treated patients (P = 0.003). The proportion of the persistent NTHi strains was similar in both groups. AZT treatment followed by adenotonsillectomy did not completely eliminate NTHi from pharynges; however, it significantly reduced the risk of carriage of Haemophilus influenzae inside the CD14(+) cells.

Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

Antimicrobial resistance and genotypes of staphylococci from bovine milk and the cowshed environment.

Investigation of antimicrobial resistance and genetic relatedness of staphylococci from milk of cows with mastitis and cowshed environment was the aim of this study. Antimicrobial resistance against 14 antimicrobials were determined by using a disc diffusion method. Genetic similarity between the most frequently isolated species was analysed by PFGE (pulsed-field gel electrophoresis). Haemolytic activity, DNase, protease and esterase production was also investigated. Coagulase-negative Staphylococcus species were isolated from 30.8% of milk samples from cows with mastitis. The most frequently isolated species was Staphylococcus xylosus and yield of these organisms was significantly associated with milk of mastitis cows. S. epidermidis was a predominant penicillin-resistant species. High frequency of resistance to lincomycin was observed among isolates of S. sciuri (54.2%) and S. xylosus (25.9%) from cows with mastitis. PFGE (pulsed-field gel electrophoresis) analysis of 29 Staphylococcus aureus isolates showed the presence of 17 PFGE pulsotypes. Isolates of S. sciuri (n = 36) had unique PFGE patterns. Some S. xylosus isolates from milk and milker's hands had the same PFGE pulsotypes, and this observation could indicate that dairyman may be a potential source of the infection. The pulsotype of each of the remaining isolates of S. xylosus suggested that they might have come from common environmental sources; however, these isolates differed in antibiotic resistance pattern or virulence traits. Therefore, knowledge about antibiotic sensitivity pattern and virulence factors of a CNS isolate, besides its genotype, may be informative in tracking the source of the infection.

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces.

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.

The application of PCR to the identification of selected virulence markers of Yersinia genus.

The subject of this study was thirty nine strains of Yersinia enterocolitica, isolated from faeces of humans who showed symptoms typical of intestinal yersiniosis, and seventy strains of Y enterocolitica, four strains of Y. pseudotuberculosis, and one strain of Y. kristensenii from healthy pigs. In the population tested the following serogroups appeared: O3, O9, O2, O5. A PCR was used to detect the presence of pathogenic chromosomal markers, such as myfA and inv genes of the tested Yersinia species. Among Y. enterocolitica strains isolated from humans and belonging to serogroup O3 (thirty four strains) and serogroup O9 (five strains) thirty three Y. enterocolitica O3 strains and four Y. enterocolifica O9 strains, gave a positive reaction to the nmyfA gene, yielding a fragment of 280 base pairs (bp). Among seventy Y. enterocolitica strains isolated from pigs forty strains belonging to serogroup O3 and fifteen strains belonging to serogroup O9 gave a positive reaction to the myfA gene. The presence of 390 bp amplified products, corresponding to the inv gene fragment, was detected in PCR products of three Y pseudotlluberculosis strains from pigs and only in one Y. enterocolitica O3 strain from humans, which had no myfA gene. The results obtained show that the myfA gene is only present in the strains that belong to pathogenic serotypes of Y. enterocolitica. The myfA gene prevailed in the Y. enterocolitica O3 and O9 strains from humans but was less common in the Y. enterocolitica O3 and O9 strains from pigs.

Antibacterial activity of liposome-encapsulated antibiotics against Pseudomonas aeruginosa attached to the matrix of human dermis.

The present studies were undertaken to compare the antibacterial activity of liposome vesicles containing amikacin, ciprofloxacin or polymyxin B in the removal of P. aeruginosa organisms from microcolonies growing on sections of the matrix of human dermis. Encapsulation efficiency of antimicrobials inside cationic liposomes was 30% for amikacin, 50% for ciprofloxacin, and 100% for polymyxin B. The sections of dermis were colonized for 72 h with P. aeruginosa strains isolated from burn wounds. After that time, an intense growth of microorganisms on the dermis surface was observed. The sessile organisms were treated (with mild shaking) with solutions of either liposomal or free amikacin, ciprofloxacin, and polymyxin B for 1 h, and also with a mixture of liposomal or free ciprofloxacin and polymyxin B (1:1) for 20 min. After treatment with liposomal antimicrobials, the mean per cent of viable cells attached to the dermis was 48.7% for liposomal amikacin, 17.4% for liposomal ciprofloxacin, 19.1% for liposomal polymyxin B, and 3.6% for a mixture of liposomal ciprofloxacin and liposomal polymyxin B. Removal of P. aeruginosa from microcolonies growing on the dermal matrix was more effective when liposomal formulations were used compared to the free antibiotics. Therefore, cleansing of the contaminated matrix of human dermis with liposomal ciprofloxacin, liposomal polymyxin B or with the mixture of both liposomal antibiotics seems to increase the efficacy at the removal of attached bacterial cells.

Antimicrobial properties of copper-coated electroconductive polyester fibers.

Three synthetic copper-coated EURO-static fibers (PET--polyester, PA--polyamide, and PAC--polyacrylamide) manufactured by EUROPA Corporation S.C., Poland, were tested as potential antimicrobial agents. The inhibitory properties of the fibers were examined using different microorganisms as follows: i. Staphylococcus aureus ATCC 25293, and Pseudomonas aeruginosa ATCC 27853 reference strains, ii. 8 strains of S. aureus (4 MRSA and 4 MSSA) and 5 strains of P. aeruginosa isolated from infected wounds, and iii. fungal pathogen Scopulariopsis sp. isolated from onychomycosis case. The results of experiments have evidenced that polyester (PET) copper-coated EURO-static fibers inhibit the growth of all the strains used.

Susceptibility of adherent organisms from Pseudomonas aeruginosa and Staphylococcus aureus strains isolated from burn wounds to antimicrobial agents.

To assess the bactericidal effects of ciprofloxacin, netilymicin, and polymyxin B on adherent Pseudomonas aeruginosa organisms and also the bactericidal effects of ciprofloxacin, vancomycin and teicoplanin on adherent Staphylococcus aureus cells, a simple end-point microplate assay, based on the method described by Miyake et al. was used in the present study. As results of the assay, the minimal inhibitory concentration (MICADH) values are taken, which express the susceptibility of the bacterial cells spontaneously released from the surface of adherent microcolonies to antimicrobial agents. Also, a minimal bactericidal concentration (MBCADH) value was read, which is defined as the lowest antibiotic concentration required to kill the sessile bacterial cells. For twenty P. aeruginosa strains and nineteen S. aureus strains isolated from burn wounds, an enhanced resistance against bactericidal action of the applied antibiotics was observed when bacterial cells were attached to polystyrene surface. The MICADH values were comparable with the conventional MIC values only for ciprofloxacin and netilmicin for P. aeruginosa strains. The MBCADH values exceeded many times the conventional MBC values for the majority of strains. The validity of the assay was estimated in the experiment designed to determine the concentration of ciprofloxacin that should be released topically from the collagen dressing to prevent the biomaterial from microbial colonization and allow the decontamination of the wound.

Adhesion of Pseudomonas aeruginosa to collagen biomaterials: effect of amikacin and ciprofloxacin on the colonization and survival of the adherent organisms.

The adherence of P. aeruginosa to collagen membrane, sponge, and to a new anti-infective COLL dressing and the susceptibility of the organisms attached to the biomaterials to amikacin were investigated in vitro. After 17 h of attachment, the bacteria demonstrated an increased resistance to amikacin compared with their free-floating counterparts. Amikacin, even at a concentration exceeding 150 times the minimal bactericidal concentration (MBC) for the strain tested, did not eradicate the attached bacteria from the surface of collagen membrane. However, when the drug at a high concentration (over 16 times the minimal inhibitory concentration, MIC) was present in the incubation medium before it had been inoculated with P. aeruginosa, a reduction of 2 log10 units in the organisms adherent to the surface of collagen membrane was observed. We conclude that slow release of the antibiotic from the COLL dressing could control the bacterial colonization on the surface. In fact, the released amikacin at the final concentration of 32 times the MBC reduced the number of adherent bacteria by 6 log10 units. In contrast, ciprofloxacin at the same final bactericidal concentration completely eradicated the bacteria from the surface of COLL dressing. However, as ciprofloxacin is not recommended for use as a topical antimicrobial agent, a further search is needed to find an agent with a similar anticolonization activity.

A new anti-infective collagen dressing containing antibiotics.

A new antibacterial dressing for infected wounds was prepared. The dressing was composed of a collagen membrane and collagen sponge; both biomaterials possess good tissue biocompatibility. An active antibacterial layer of limited hydrophobicity was placed between the membrane and the sponge and into the upper part of the sponge. The dressing contained gentamycin or amikacin at concentrations of 0.3 microgram/cm2 (loading level of the drug utilized during preparation of the dressing). Either the antibiotic or its concentration easily can be changed in the dressing by the manufacturer. The dressing was stable for several months. The antibiotic was released slowly from the dressing in in vitro experiments for 3 days. Antibacterial activity of the dressing was tested using a mouse wound model experimentally infected with Pseudomonas aeruginosa. Both dressings, containing either amikacin or gentamycin, reduced the number of living bacterial cells in the infected tissue almost to zero during the course of observation. The new dressing may be effective in the treatment of infected wounds in patients.

Release of antibiotics from collagen dressing.

Our new collagen dressing has been developed recently. Three types (A, B, and C) of the dressing were prepared in this study. Each type contained bacitracin, neomycin or colistin. The antibiotic was input into: i. collagen sponge (CS)--type A, ii. layer of limited hydrophobicity (LLH)--type B, and iii. into both CS and LLH layers--type C. The final concentration of the antibiotic that resulted from the loading level was 2 mg/cm2 for the dressings of type A and B and 4 mg/cm2 for the dressing of type C. The antibiotics were then extracted from the pieces of dressings for two days through dialysis membrane. Susceptibility of 54 bacterial strains (S. aureus, P. aeruginosa, and Acinetobacter) isolated from burn wounds were tested to the three antibiotics used for preparation of the dressings. The results of the study evidenced that efficiency of released of antibiotics into the extracts depended on the kind of antibiotic and on the type of dressing. The concentration of the antibiotics proved to be much higher than MIC90 values of the bacterial isolates tested in respect to their susceptibility. The dressing containing mixture of the three antibiotics in two layers--CS and LLH is now considered as potentially effective for care of infected wounds. It may be useful for the treatment of infected wounds or for profilaxis of contaminated wounds, ensuring: i. sufficient antimicrobial activity in wound, and ii. optimal wound environment for the presence of collagenic biomaterial on the damaged tissue.

A simple in vitro model to test the efficacy of antimicrobial agents released from dressings.

A new model for in vitro evaluation of an activity of antibacterial agents released from delivery systems containing antibiotics or antiseptics was developed. The model was composed of two layers of agar gel of two different concentrations in PBS. In the upper gel, the wells were cut and filled with polyurethane sponges saturated with bacterial broth culture. The sponges were then covered with tested dressings containing antibacterial agent. The model was tested by using two bacterial strains, Pseudomonas aeruginosa or Staphylococcus aureus and experimental collagen dressing with doxycyclin, amikacin, or with silver sulfadiazine. The number of colony-forming units (CFU) in the polyurethane sponges was significantly lower under the dressings with antibacterial agents as compared to the control (the same dressing without antimicrobials) during 3 days of observation. The new in vitro model can be recommended for examination of different antibacterial delivery systems instead of experimentally infected wounds in laboratory animals or instead of the other in vitro models.

Anti-pseudomonal activity of collagen sponge with liposomal polymyxin B.

An antibiotic delivery system has been developed using collagen sponge with liposome-encapsulated polymyxin B. Superficial, non-lethal infection was produced in mice by injecting Pseudomonas aeruginosa into the skin windows. Wounds were dressed with collagen sponge containing liposomal polymyxin B or containing empty liposomes (with PBS) as a control. Single dose of topically applied collagen sponge with encapsulated polymyxin B decreased bacterial cell number as compared to the control. Finally, after 8 days of experiment, the number of bacterial colonies dropped below 10(4) per biopsy. Presented polymyxin B delivery system offers potential clinical advantages.

The influence of Pseudomonas aeruginosa on liposomes.

The interaction of liposomes loaded with Ponceau red (used as a marker) with Pseudomonas aeruginosa cells was observed and it resulted in marker leakage. The marker leakage from liposomes was low in physiological fluids. The interaction was independent of secreted phospholipase C level and the serotype of the tested strain. Six of 37 examined isolates did not cause any release of the marker from the liposomes. Marker release of over 50% of total encapsulated material was observed only for ten of the strains tested.

Effects of free and liposome-encapsulated antibiotics on adherence of Pseudomonas aeruginosa to collagen type I.

The adherence of 27 clinical Pseudomonas aeruginosa strains to collagen type I was investigated by using a solid-phase assay. The influence of free antibiotics (amikacin, gentamicin, piperacillin, bacitracin, and polymyxin B) and liposome-entrapped antibiotics (amikacin and polymyxin B) on bacterial attachment to collagen type I was examined. The greatest inhibitory effect was shown for free and liposomal amikacin, which decreased the attachment of 74 and 100% of tested strains, respectively. The mean percent attachment (+/- standard deviation) in the presence of free amikacin was 65.7% (+/- 12.0%) as measured by solid-phase assay. In the presence of liposomal amikacin, the attachment ranged from 17.3% (+/- 6.0%) to 42.1% (+/- 9.4%), depending on the antibiotic solvent. In contrast, polymyxin B, even at a subinhibitory concentration, enhanced attachment of all P. aeruginosa isolates to collagen. Liposomal polymyxin B displayed a protective effect only when the encapsulated drug was of a low concentration. Application of liposome-encapsulated amikacin may be advantageous in injured tissues in which extracellular matrix structures become exposed.

Modulation of Pseudomonas aeruginosa adherence to collagen type I and type II by carbohydrates.

This study was undertaken to examine if receptor recognizing saccharides may be involved in the adherence of Pseudomonas aeruginosa to collagen type I and type II. We performed an adherence inhibition assay: cells of individual P. aeruginosa isolates attached to immobilized collagen type I or type II in the presence of monosaccharides, which could serve as blockers of bacterial receptors. Bacterial binding to collagen type I molecules was inhibited to the highest degree by sugar composition D-galactose/D-mannose/N-acetylneuraminic acid (5:5:1), whereas attachment of P. aeruginosa to collagen type II was inhibited by composition d-glucose/D-galactose (1:1). The same strains which were sensitive to inhibition of binding to collagen type II by both collagen types, were also sensitive to blocking by composition D-glucose/D-galactose. It suggests that saccharides play a role in adherence of P. aeruginosa to collagen type I and type II, and a common receptor for both types of collagen may be available on the surface of P. aeruginosa cells.

Antibacterial activity of liposomal amikacin against Pseudomonas aeruginosa in vitro.

The influence of liposomal amikacin on Pseudomonas aeruginosa was studied. P. aeruginosa clinical isolates caused release of encapsulated amikacin from liposomes. The liposomal amikacin proved to be active as bactericidal agent after 3 h of incubation with P. aeruginosa. Incubation of P. aeruginosa with liposomal amikacin resulted in inhibition of the growth when equivalent of 2 MIC was added but not when equivalent of 1 MIC was added. Susceptibility of bacterial isolates to the liposomal amikacin varied with bacterial strain used, but generally encapsulation of amikacin did not enhance their antibacterial activity.

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

2.2 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor.

. The crystal structure of a ternary complex containing the catalytic subunit of cAMP-dependent protein kinase, ATP and a 20-residue inhibitor peptide was refined at a resolution of 2.2 A to an R value of 0.177. In order to identify the metal binding sites, the crystals, originally grown in the presence of low concentrations of Mg(2+), were soaked in Mn(2+). Two Mn(2+) ions were identified using an anomalous Fourier map. One Mn(2+) ion bridges the gamma- and beta-phosphates and interacts with Asp184 and two water molecules. The second Mn(2+) ion interacts with the side chains of Asn171 and Asp l84 as well as with a water molecule. Modeling a serine into the P site of the inhibitor peptide suggests a mechanism for phosphotransfer.

Characterization of antibody response to Pseudomonas aeruginosa in patients with wound infections.

ELISA was used to measure the amount and avidity of IgG antibodies to exotoxin A (ExA) and 7 Fisher's immunotypes of lipopolysaccharide (LPS) in the sera of 13 patients with mild or moderate Pseudomonas aeruginosa infections. Changes in the specificity of tested sera during the course of infection were demonstrated. A statistically significant increase was seen in the amount and avidity of the antibodies to ExA in a majority of the sera, and an increase was seen in amount of antibodies to LPS immunotype 4 in the sera of patients with moderate infections.