The strongest resistance of Staphylococcus aureus to erythromycin is caused by decreasing uptake of the antibiotic into the cells.

The consequence of excessive use of macrolides is a high occurrence of mechanisms responsible for resistance to these drugs. Of 97 erythromycin-resistant bacterial strains gathered in the Wroclaw area in Poland, 60% exhibited very high resistance, and those with the inducible MLS(B) (macrolide-lincosamide-streptogramin B) resistance phenotype predominated. Direct genetic investigation revealed that the erm genes coding for ribosomal methylases are the most frequently occurring erythromycin resistance-determining genes. No genetic resistance determinant was detected in 13% of the erythromycin-resistant strains. The efflux mechanism occurs in strains isolated from the nasopharyngeal cavity twice as often as in those isolated from other material, where the mechanism connected with target site modification predominates. Measurements of radiolabelled antibiotic accumulation inside bacterial cells revealed that in highly resistant strains (MIC > 1024 µg/ml), an important factor responsible for the resistance is the permeability barrier at the cell wall level. This would be a hitherto unknown mechanism of resistance to erythromycin in Staphylococcus aureus.

Penetration of a selected antibiotic and antiseptic into a biofilm formed on orthopedic steel implants.

BACKGROUND: The aim of the study was to determine the impact of octenidine hydrochloride and gentamicin on bacterial survival and reduction of biofilms formed on orthopaedic metal implants. MATERIAL AND METHODS: We studied metal orthopaedic components (screws, nails, fragments of wires used in Ilizarov devices) and a bone sequester. The presence and intensity of biofilm formation on the medical biomaterials was determined using the method of Richards et al. by visual evaluation of 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by viable bacteria. The presence and structure of the biofilm on the components of the Ilizarov device, screws and bone sequester was also studied by electron microscopy. Bacterial survival in the biofilm following exposure to the antibiotic and antiseptic was studied by CLSI microdilution method in microtitre plates using TTC. Results. Most of the 16 strains (S. aureus, S. epidermidis, E. coli, Enterobacter) isolated from orthopaedic implants were able to form a biofilm. Established biofilms were resistant to gentamicin and octenidine hydrochloride but demonstrated greater susceptibility to octenidine. CONCLUSIONS: The results of the study indicate that octenidine hydrochloride is more effective than gentamicin in the treatment of infections associated with the formation of a biofilm on orthopaedic implants.

[Antibodies to Klebsiella O-antigens in patients with seronegative spondyloarthropaties]. / Przeciwciala przeciwko antygenom somatycznym O paleczek Gram-ujemnych z rodzaju Klebsiella u chorych na spondyloartropatie seronegatywne.

OBJECTIVES: The pathogenesis of seronegative spondyloarthropathies is still unknown. A microbial etiology has been suggested. The aim of the study was to analyze the antibodies against Klebsiella O-antigens in serum of patients with seronegative spondyloarthropathies. METHODS: 30 patients with seronegative spondyloarthropathies, 20 with rheumatoid arthritis and 20 healthy volunteers were included in the study. The serum antibodies against Klebsiella O1 and O3 antigens were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: In serum of patients with seronegative spondyloarthropaties the antibodies against Klebsiella antigen O1 and O3 occur less frequently (6.67%) than in serum of patients with rheumatoid arthritis (35%) and that in serum of healthy subjects (40%). CONCLUSIONS: The results do not confirm the role of LPS in the pathogenesis of seronegative spondyloarthropthies, but on the other hand we could not exclude the concept that it may play an important role.

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization.

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.

[Adhesion dependence of coagulase-negative staphylococci to biomaterials and PIA polysaccharide synthesis on icaADBC operon presence]. / Zaleznosc adhezji gronkowców koagulazo-ujemnych do biomaterialów oraz syntezy polisacharydu PIA od obecnosci operonu icaADBC.

Both Staphylococcus epidermidis and Staphylococcus haemolyticus are important causes of infections associated with catheters and other medical devices. This infections result in significant morbidity, mortality and economic cost. It has recently been shown that not only S. epidermidis but also S. haemolyticus can produce slime and carries the ica operon responsible for and slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. This study is focused on detecting icaA and icaD genes in S. haemolyticus and comparison of these two species. It turned out that strain representatives within the same species behave very differently and a single tested strain from each species is unlikely to be representative of the species as a whole. Contrary to S. epidermidis, S. haemolyticus strain appeared to carry no icaA-like and icaD-like genes, but was able to form biofilm in vitro.

[Risk of coagulase-negative staphylococci infections in catheterized patients due to adhesion and slime production]. / Ryzyko zakazenia gronkowcami koagulazoujemnymi u cewnikowanych chorych zwiazane z adhezja i wytwarzaniem sluzu.

UNLABELLED: Coagulase-negative staphylococci are important causes of infections associated with biomaterials, e.g. catheters. Aim of the study was to determine pathogenic traits of S. epidermidis: slime producing and adhesion to biomaterials. MATERIALS AND METHODS: The researches have been done on S. epidermidis strains isolated from newborns hospitalized on BCU. Defining an ability of slime production was done by means plate method--according to Christensen. Adhesive abilities of the strains to biomaterials were analyzed by means of Richard's and co. method which consist in estimating the level of a substrates's reduction--TTC (2,3,5-triphenyltetrazolium chloride) to an insoluble formazan. RESULTS: Among 41 S. epidermidis strains 68% labeled as slime producing and 32% as non-producing. Most of slime producing strains showed reduction value of TTC at +4 and +3. All non-producing S. epidermidis strains showed reduction value of TTC at 2+. CONCLUSION: The strains producing slime displayed greater adhesive abilities in comparison to the non-producing ones.

[Occurrence and characteristic of P-like adhesin among Klebsiella strains]. / Wystepowanie i charakterystyka powierzchniowego bialka P-like paleczek Klebsiella.

Colonisation and remaining of microorganism on mucus membrane of microorganism is tightly connected with adhesion mechanisms and determine the first step of physiological settlement of the organism or the first stage of clinically demonstrated infection. In Klebsiella rods there are known three types of fimbrial adhesins (type 1, 3 and KPF-28) and non-fimbrial adhesin CF29K. It is stated that Klebsiella strains adhesions are responsible for their adherence to the epithelial cells of both respiratory and urinary tracts and to intestine epithelium. The in vitro research affirmed Klebsiella rods adherence to protein matrix. The aim of our work was the establishment of character, receptor specificity and the appearance frequency of P-like called adhesin. The frequency of expression of P-like adhesin was estimated among 380 isolated from the patients strains on the basis of agglutinating methods. The amorphic character of P-like adhesin was proved using electron microscopy method. The isolation and purification of P-like protein with a help of affinity chromatography enabled to estimate the receptor specificity of the adhesin. The receptor specificity was established as similar to E.coli PapG adhesin.

Helicobacter pylori antigens in stool specimens of gastritis children before and after treatment.

BACKGROUND: Various testing methods are successfully applied to the diagnosis of Helicobacter pylori infection, but noninvasive techniques are still needed for therapeutic monitoring, especially in children. In the search for new noninvasive techniques for the diagnosis of H. pylori infection, the authors evaluated an enzyme immunoassay for the detection of H. pylori antigen in stool (HpSA). METHODS: The authors studied 62 H. pylori-positive children with chronic gastritis and 45 control subjects. H. pylori infection was diagnosed using cultures and histology of gastric biopsy specimens and a stool antigen test before treatment (clarithromycin, amoxicillin, omeprazole for 7 days) and 4 weeks to 6 weeks after treatment. RESULTS: Before therapy, antigen in stool was detected in 55 of 62 H. pylori-positive patients, which indicates that the sensitivity of the HpSA test was 88.7%. Of the 45 control subjects (with negative culture and histology results), 43 had negative results for H. pylori in the stool test (specificity, 95.5%). After completion of therapy, eradication was obtained (and confirmed by culture and histology) in 53 of the 62 H. pylori-positive children (85.5%). Four weeks to 6 weeks after eradication therapy, the sensitivity, specificity, positive predictive value, and negative predictive value of the stool antigen (HpSA) test were 88.9%, 96.2%, 80%, and 98%, respectively. CONCLUSIONS: The accuracy of the HpSA test for the detection of H. pylori in human stool 4 weeks to 6 weeks after treatment is comparable with the accuracy of the culture results. The stool antigen (HpSA) test was found to be a useful method for posttreatment eradication testing of infection in children.

[Selected features determine pathogenicity of Staphylococcus haemolyticus]. / Wybrane cechy warunkujace chorobotwórczosc Staphylococcus haemolyticus.

The study have been done on S. haemolyticus strains isolated from patients hospitalized an Surgical Unit. Aim of the study was to determine pathogenic traits of S. haemolyticus: slime producing, adhesion to biomaterials, antibiotics susceptibility and the profiles of surface proteins. Among 44 S. haemolyticus strains, in the test-tube method, there have been 38% labeled as slime producing and 62% as non-producing. In the plate method at 48% slime production was noticed, while 52% strains did not produce slime. It is quite significant that all CNS strains which have an ability to produce mucus, that was proved by means of two methods (test-tube and plate), show a high level of TTC's reduction to formazan. The analysis of resistance to antibiotics in relation to slime production demonstrated more frequent antibiotic resistance of the slime-producing strains.

[Adhesive properties and antibiotic resistance of Klebsiella strains isolated from gastrointestinal tracts of children hospitalised in Wroclaw nad Opole]. / Wlasciwosci adhezyjne i lekoopornosc szczepów Klebsiella izolowanych z przewodu pokarmowego dzieci hospitalizowanych we Wroclawiu i w Opolu.

Gram negative Klebsiella bacilli present many pathogenic properties, which determine their ability to survive and rapid spreading in hospital environment. There are many factors responsible for the pathogenicity of Klebsiella strains: capsule, fimbriae, nonfimbrial adhesins, lipopolysaccharide of the cell wall and extracellular secreted exotoxins. Klebsiella strains are etiological agents of different nosocomial infections but also colonized gastrointestinal and respiratory tracts. The aim of our work were adhesive properties and antibiotic resistance of Klebsiella strains isolated from stool of hospitalized children, according to source of potential nosocomial infections--100 Klebsiella strains from Wroclaw and 76 strains from Opole, isolated in cases of diarrhea. The resistance of this strains to different group of antibiotics, the expression of ESBL enzymes, the activity in hemagglutination and their ability to adherence to different cell lines were tested. The highest resistance of all strains to aminopenicillins was observed. The production of ESBL was highest in strains from Opole (51% strains) then in Wroclaw (9%). In both hospital units, ESBL+ strains were resistant to aminoglicosides and cotrimoxazol but sensitive to ciprofloxacine. Using hemagglutination method the types of fimbriae were defined. Above 90% investigated Klebsiella strains showed the presence of fimbriae (in Wroclaw more strains simultaneously expressed fimbriae type 1 and 3, in Opole mainly fimbriae type 3). Over 70% strains demonstrated the high level of adherence to cell lines. Only several strains showed the low level or the lack of adhesion. These results suggested that among Klebsiella strains in gastrointestinal tract were presented multiresistant strains with high ability to adherence, which may be potential source of nosocomial infections.

[Siderophores and hemolysins in iron aquisition by Acinetobacter genus]. / Siderofory i hemolizyny w pozyskiwaniu zelaza przez paleczki z rodzaju Acinetobacter.

In 60 strains of Acinetobacter genus isolated from clinical material belonging to species A. baumannii, A. haemolyticus, A. junii and A. lwoffii a hydroxamate and phenol-catechol class siderophores was identified by chemical and biological testes. A correlation between siderophores production and growth intensity, species affiliation and origin of strains was found.

[The effect of culture conditions on expression of surface proteins from coagulase-negative staphylococci isolated from clinical material of newborns hospitalized in an intensive care unit]. / Wplyw warunków hodowli na ekspresje powierzchniowych bialek gronkowców koagulazoujemnych izolowanych z materialu klinicznego od noworodków hospitalizowanych na oddziale intensywnej terapii.

A surface adhesion is a fundamental stage, in a pathogenesis of inflammations caused by coagulase-negative staphylococci, at which surface proteins take part. The proteins that were found at S. saprophyticus and ascribed to take part in polystiren plates adhesion: SSP-1 and SSP-2 could be an example. In this work the effort to esteem surface protein expression of various kinds of coagulase-negative staphylococcus according to cultivation conditions has been made. The studies carried out with 31 staphylococcus strains being obtained from new-born children hospitalised in Neonatology Clinic helped to analyse the similarities among these proteins in the area of a given strain cultivated on different mediums. The examined strains belonged to five different species and it had been taken into consideration during the results esteem. On the grounds of electrophoretic surface proteins division the charts dysometric analysis was created showing interdependence between Rf for surface proteins and optical density. Afterwards an analysis for each strain was done taking into account a sort of medium on which the cultivation had been placed. S. haemolyticus strains analysis cultivated on three different mediums allows to isolate two protein groups showing similar expression of both low- and high-molecular proteins. For the majority of strains belonging to the same species one can observe a similar expression within low- and high-molecular surface proteins regardless of cultivation conditions. Recurrence of the electrophoretic pictures regardless of changes in cultivation conditions, creates the base for the recognised proteins identification and also for the proteins with unmarked activity isolation.

[Appearance of Helicobacter pylori antigen in stool specimens of children with chronic gastritis]. / Wystepowanie antygenów Helicobacter pylori w kale dzieci chorych na przewlekle zapalenie blony sluzowej zoladka.

Various examinations are successfully applied for the diagnosis of Helicobacter pylori infection, but non-invasive techniques are still required for the therapeutic monitoring after eradication therapy, especially in children. In the search for new non-invasive techniques to diagnose H. pylori infection, we evaluated an EIA for H. pylori antigen in stool (HpSA). Our study included 62 H. pylori-positive children with chronic gastritis. H. pylori infection was diagnosed by using the culture of gastric mucosa before treatment (clarithromycin, amoxicillin, omeprazole for 7 days) and after 3 months following treatment. Before therapy, stool antigen was detected in 55 out of 62 H. pylori-positive patients, which indicates that the sensitivity of the test was 88.7%. After therapy, eradication was obtained (and confirmed by culture) in 53 out of 62 subjects. When the HpSA test was used, the ratio was 52 out of 62. The sensitivity, specificity of HpSA was 88.9% and 96.2, respectively. The accuracy of the HpSA test for the detection of H. pylori in human stool 3 months after treatment is comparable with the accuracy of the culture results.

Characterization of coagulase-negative staphylococci by primer-specific polymerase chain reaction and ribotyping.

OBJECTIVE: To analyze, by primer-specific polymerase chain reaction (PCR) and ribotyping, coagulase-negative staphylococci (CNS). METHODS: Forty-five clinical isolates of CNS were identified by the API ID32 STAPH system and ribotyping. Additionally, primer-specific PCR was evaluated for identification of clinical strains of Staphylococcus epidermidis. RESULTS: Forty-five isolates of CNS from neonates with nosocomial bacteremia were studied. The results of the S. epidermidis-specific PCR were compared with those obtained using ribotyping and the API ID32 STAPH system. Excellent congruence was found between primer-specific PCR and ribotyping. Primer-specific PCR proved to be a fast and reliable method for the identification of S. epidermidis strains. According to the primer-specific PCR and ribotyping analysis, a few CNS isolates were found to be incorrectly identified by the API ID32 STAPH system. CONCLUSIONS: Primer-specific PCR is a fast and reliable method for the identification of S. epidermidis. Primer-specific PCR in combination with ribotyping is a promising approach for studying the epidemiology of S. epidermidis and other CNS species in hospital.