Therapeutic potential of bacteriophages for staphylococcal infections and selected methods for in vitro susceptibility testing of staphylococci.

AIM: Staphylococcus aureus strains are the cause of frightening hospital and community infections, especially when they are resistant to antimicrobials, have important pathogenicity factors, or have biofilm production ability. Looking for novel therapeutic options which would be effective against such strains is one of the highest priorities of medicine and medical research. The study aim was to describe the occurrence of S. aureus strains and proportion of methicillin resistant strains (MRSA) detected in laboratories of the Microbiological Institute, Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno in 2011-2018. Selected strains of S. aureus were tested for biofilm production ability and susceptibility to antimicrobials and Stafal®, a phage therapeutic agent. A prerequisite was to develop a simple routine method suitable for phage susceptibility testing of bacteria. MATERIAL AND METHODS: Altogether 867 clinical isolates of S. aureus and 132 strains of other species of the genus Staphylococcus (isolated in 2011-2017) were tested for susceptibility to the phage therapy preparation Stafal® using the double-layer agar method. All strains of S. aureus were tested for biofilm production ability by the modified Christensen method with the use of titration microplates and for susceptibility to antistaphylococcal antibiotics by the disk diffusion test. For 95 S. aureus strains, the outcome of the double-layer agar method (DAM) was compared with that of our newly designed method (ODM) based on optical density decrease of the bacterial suspension. RESULTS: During the study period, the laboratories of the Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno detected 2900 strains of S. aureus per year on average. The proportion of MRSA among S. aureus isolates from blood culture and venous catheters ranged between 8.8-15.2 %. S. aureus strains recovered from venous catheters and blood culture were confirmed as stronger biofilm producers than those from other clinical specimens. MRSA strains showed higher biofilm production than methicillin susceptible strains (MSSA). As many as 90.4 % of S. aureus strains tested susceptible to the Stafal® preparation. Even a higher proportion, i.e. 99.0 %, of MRSA strains were Stafal® susceptible. No relationship was found between Stafal® susceptibility and biofilm production ability. Although Stafal® targets primarily S. aureus, some susceptibility (26.5 %) was also found for other staphylococcal species. A novel simple method designed for routine testing of susceptibility to phage therapy preparations based on optical density decrease was comparably sensitive and reliable as the commonly used double-layer agar method (DAM) and, in addition to being easy and rapid to perform, after prolonged suspension culture and at higher measurement frequency, it has an extra advantage of providing the possibility for monitoring also phage action dynamics. CONCLUSIONS: The proportion of MRSA strains detected in this study is comparable to that reported for the whole Czech Republic, and the biofilm production data are consistent with scientific evidence. The host range of the Stafal® preparation is relatively wide and covers most strains of S. aureus and some coagulase negative staphylococci. The highest efficiency of Stafal® (99.4 %) was observed against MRSA strains with multiple types of antibiotic resistance. In vitro testing of 867 strains of S. aureus and 132 other staphylococcal species has shown the phage therapy preparation Stafal® to be a suitable candidate therapeutic option for the treatment of staphylococcal infections, especially in case of failure of conventional antibiotic therapy. Moreover, a simple method for routine phage susceptibility testing of clinical bacterial isolates has been designed, which is an essential tool to be used in phage therapy.

Antimicrobial effect of novel hydrogel matrix based on natural polysaccharide Sterculia urens.

INTRODUCTION: Materials for modern wound-management are a very broad and heterogeneous group. One of the most important representatives is natural materials, or more precisely polysaccharides isolated from various plants and animals. With the increasing resistance of pathogens to established antimicrobial agents, there is also an attempt to discover new mechanisms of the effects of these materials. Gum karaya (GK) is a very promising representative of the natural polysaccharides group and, since it is obtained from Sterculia urens as resin, it is also possible to assume its certain antimicrobial activity. MATERIAL AND METHODOLOGY: The antimicrobial potential of GK and chitosan (Ch) has been tested on several preselected strains to match the real epidemiological situation of the agents of infectious complications in the field of burned wounds. Tested strains included representatives of gram-positive and gram-negative bacteria as well as selected yeasts. Methicillin susceptible Staphylococcus aureus CCM 4223 (ATCC 29213), methicillin resistant Staphylococcus aureus CCM 4750 (ATCC 43300), Klebsiella pneumoniae CCM 4985 (ATCC 700603), Candida albicans CCM 8261 (ATCC 90028), Pseudomonas aeruginosa CCM 3955 (ATCC 27853) were obtained from the Czech Collection of Microorganisms. Pseudomonas aeruginosa FF 1, Pseudomonas aeruginosa FF 2 and Pseudomonas aeruginosa FF 3 (all multi-resistant clinical strains), Staphylococcus epidermidis A 013, Staphylococcus epidermidis A 117, and Candida parapsilosis BC 11 were obtained from the Collection of Microorganisms at the St. Annes University Hospital, Brno. Antimicrobial tests were performed using the disk diffusion test methodology. Another set of antimicrobial tests was obtained by measuring the growth curves. RESULTS: Bacteriostatic activity testing showed 1% GK concentration and both 1% and 0.5% chitosan concentration effective against all pathogens tested. The combination of GK50/Ch50 in concentrations of 1% and 0.5% had similar or better effect. Lower concentrations of the combined material are poorly effective against tested strains. Bactericidal activity testing has not produced positive results, except for Candida spp., where only a partial effect of GK50/Ch50 was observed at 1% concentration. In the growth curve test, the efficiency of both GK alone and chitosan was found to be significantly higher in gram-positive bacteria compared to gram-negative ones. In the case of this experiment, only a one-tenth concentration was used compared to the disk diffusion test concentration. This results correspond with the data from the bacteriostatic activity testing. CONCLUSION: This is the first publication that attempts to comprehensively define the potential for GK antimicrobial activity and also the possible potentiation of this activity with the use of chitosan. Further experiments are needed to extend the antimicrobial efficiency to gram-negative bacteria.

High frequency of Candida fabianii among clinical isolates biochemically identified as Candida pelliculosa and Candida utilis.

Clinical yeast isolates belonging to Candida pelliculosa, Candida utilis and Candida fabianii are difficult to distinguish in a routine mycology laboratory using common biochemical tests. The aims of this study were to determine the prevalence of C. pelliculosa, C. utilis and C. fabianii in clinical samples and to compare their minimum inhibitory concentrations (MICs) to systemic antifungals. Two hundred and forty-eight clinical yeast isolates obtained from eight large hospitals in the Czech Republic were included in this study. Identification was performed biochemically using ID 32C kit and by MALDI-TOF MS. MICs were determined using colorimetric broth dilution Sensititre YeastOne panels. From a total number of 248 isolates, 175 were identified as C. pelliculosa and 73 as C. utilis using the biochemical kit. In contrast, MALDI-TOF MS identified 222 isolates as C. fabianii, 20 as C. pelliculosa and 6 as C. utilis. The highest mean MICs were found in C. fabianii and, regardless of the studied species, in isolates from blood cultures and central venous catheters. MALDI-TOF MS revealed C. fabianii to be most prevalent in clinical samples as compared with the other studied species. Higher MIC values in C. fabianii support the importance of correct identification of this species.

[Alloplastic Materials and their Propensity to Bacterial Colonisation]. / Aloplastické materiály z pohledu jejich citlivosti ke kolonizaci bakteriemi.

UNLABELLED: PURPOSE OF THE STUDY The alloplastic materials currently used for protective surface layers on implants were tested in vitro under microbiological laboratory conditions by contamination with microbial agents most frequently found in deep infection of total joint replacements. The objective was to find out how the resistance to bacterial colonisation was related to different surface finishes. MATERIAL AND METHODS Each of 14 samples of alloplastic material currently used in the manufacture of orthopaedic implants was inoculated with each of the group of microorganisms most frequently infecting joint replacements; these were Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli. At 24 hours of incubation, biofilms produced on sample surfaces were collected, stained with crystalline violet and assessed by spectrophotometry. The average value of biofilm absorbances (AV595) for the group of microorganism tested was taken as a basic characteristic of each material sample indicating its sensitivity to bacterial. RESULTS Of the metal materials with smooth surface finish, Vitalium (AV595, 0.368) showed the lowest affinity to microbial colonisation; next was titanium (AV595, 0.459) and steel (AV595, 0.505). A significant increase in sensitivity to bacterial colonisation was recorded in all types of surface finish of steel (AV595, 0.571) and in titanium alloy with a rough surface texture (AV595, 0.737 to 1.676); p < 0.05. Porous titanium surfaces significantly increased material affinity to colonisation. DISCUSSION Our study had certain limitations concerning in vitro evaluation of porous surfaces that have high affinity to bacterial colonisation. Porous titanium, and its hydroxyapatite layer in particular, considerably promotes osteoblast colonisation of the surface as well as implant osseointegration in the bone bed. Microorganisms therefore have no room for surface colonisation. Problematic may remain the surface parts outside contact with bone that keep their affinity to bacterial colonisation. CONCLUSIONS The material of choice for cemented implants is Vitalium which, of all metal surfaces, has the lowest sensitivity to bacterial colonisation. The materials of choice for cementless implants are titanium alloys. However, an osteoactive surface not in contact with bone remains a problem. On the one hand, its roughness and porosity are crucial to good osseointegration, on the other hand, its affinity to bacterial colonisation is high. KEY WORDS: alloplastic material, biofilm, joint replacement infection.

[Candida dubliniensis in clinical specimens and possibilities for identification]. / Výskyt Candida dubliniensis v klinickém materiálu a moznosti její identifikace.

STUDY OBJECTIVE: The species Candida dubliniensis shares a wide range of phenotypic characteristics with Candida albicans, the most common yeast species isolated from clinical specimens. This is a considerable complication for the detection and identification of Candida dubliniensis from clinical specimens. The lack of data on the incidence of C. dubliniensis in the Czech Republic was the motivation behind the efforts to detect this pathogen in specimens analyzed at the Institute for Microbiology, Faculty of Medicine Masaryk University and St. Anne's Faculty Hospital in Brno. Another aim was to test the reliability of the culture methods used. MATERIAL AND METHODS: Altogether 2260 yeast isolates initially identified as C. albicans were analysed. To differentiate C. dubliniensis from C. albicans, four phenotypic methods were used: colour-based differentiation on CHROMagar Candida medium, culture on medium with 6.5% of NaCl, growth at 42 °C, and colony characteristics on Staib agar. To verify the results, the Bichro-Dubli Fumouze latex agglutination test and species-specific polymerase chain reactions (PCR) were used. RESULTS: Using phenotypic methods, latex agglutination, and PCR, 50 (2.2%) strains from the study set were assigned to C. dubliniensis. Most (31) C. dubliniensis isolates were recovered from the respiratory tract and the remaining others were three urine isolates, four stool isolates, one central venous catheter isolate, and one blood isolate. With the exception of colour-based differentiation on CHROMagar Candida medium showing a specificity of 85.5%, all the culture methods used have a high sensitivity and a high specificity. CONCLUSION: Identification of C. dubliniensis as C. albicans was confirmed in various clinical specimens, most often from the upper respiratory tract. The colour-based differentiation of C. dubliniensis from C. albicans on chromogenic CHROMagar Candida medium can only be recommended as a screening test for the differentiation of C. dubliniensis from other species of the genus Candida. The remaining three methods are highly reliable. The final identification should be based on a combination of these methods, with the species-specific PCR or latex agglutination test used for verification.

Phage therapy combined with Gum Karaya injectable hydrogels for treatment of methicillin-resistant Staphylococcus aureus deep wound infection in a porcine model.

Skin and soft tissue infections (SSTIs) represent a significant healthcare challenge, particularly in the context of increasing antibiotic resistance. This study investigates the efficacy of a novel therapeutic approach combining bacteriophage (phage) therapy with a gum Karaya (GK)-based hydrogel delivery system in a porcine model of deep staphylococcal SSTIs. The study exploits the lytic activity and safety of the Staphylococcus phage 812K1/420 of the Kayvirus genus, which is active against methicillin-resistant Staphylococcus aureus (MRSA). The GK injectable hydrogels and hydrogel films, developed by our research group, serve as effective, non-toxic, and easy-to-apply delivery systems, supporting moist wound healing and re-epithelialization. In the porcine model, the combined treatment showed asynergistic effect, leading to a significant reduction in bacterial load (2.5 log CFU/gram of tissue) within one week. Local signs of inflammation were significantly reduced by day 8, with clear evidence of re-epithelialization and wound contraction. Importantly, no adverse effects of the GK-based delivery system were observed throughout the study. The results highlight the potential of this innovative therapeutic approach to effectively treat deep staphylococcal SSTIs, providing a promising avenue for further research and clinical application in the field of infections caused by antibiotic-resistant bacteria.

Value of PCR in surgically treated patients with staphylococcal infective endocarditis: a 4-year retrospective study.

The aim of the study was to establish a diagnostic value for broad-range polymerase chain reaction (br-PCR) and staphylococci-specific multiplex PCR (ssm-PCR) performed on surgical material from patients with staphylococcal infective endocarditis (IE). Data were analysed retrospectively from 60 patients with suspected staphylococcal IE and 59 controls who were surgically treated at three cardiosurgery centres over 4 years. Both PCR tests showed high agreement and could be aggregated. In patients with definite and rejected IE, the clinical sensitivity and specificity of PCR reached 89 and 95%, respectively. Tissue culture (TC) and PCR agreed with blood culture (BC) in 29% and 67% of IE cases. TC helped to determine aetiology in five BC negative cases while PCR aided in nine cases. Out of 52 patients with conclusive staphylococcal IE, 40 were diagnosed with S. aureus and 12 with coagulase-negative staphylococci. PCR was shown to be highly superior to TC in confirming preoperative diagnosis of IE. In addition to aid in culture negative patients, PCR helped to establish or refine aetiology in inconclusive cases. We suggest that simultaneous br-PCR and ssm-PCR performed on surgical material together with histopathology could significantly increase the performance of current Duke criteria.

[The use of molecular genetics techniques in clinical microbiology--final report from the workshop of the Molecular Microbiology Working Group TIDE]. / Aplikace metod molekulární genetiky v klinické mikrobiologii: zpráva z jednání Pracovní skupiny molekulárni mikrobiologie--TIDE.

In the last decade, there has been a rapid development in the use of molecular genetics methods in clinical microbiology. Novel technologies bring new knowledge and approaches to various disciplines of microbiology--taxonomy, identification of microbes, clinical diagnosis, epidemiology of infectious diseases and antibiotic resistance. This article summarizes the conclusions from the workshop of the Molecular Microbiology Working Group TIDE held during the Second Annual Meeting of the Society for Medical Microbiology of the J. E. Purkyne Czech Medical Association.

[Detection of viable metabolically active yeast cells using a colorimetric assay]. / Prukaz zivotaschopných, metabolicky aktivních kvasinkových bunek pomocí kolorimetrického média.

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

[Urinary catheter biofilm infections]. / Biofilmové infekce mocových katétru.

Urinary tract infections, most of which are biofilm infections in catheterized patients, account for more than 40% of hospital infections. Bacterial colonization of the urinary tract and catheters causes not only infection but also other complications such as catheter blockage by bacterial encrustation, urolithiasis and pyelonephritis. About 50% of long-term catheterized patients face urinary flow obstruction due to catheter encrustation, but no measure is currently available to prevent it. Encrustation has been known either to result from metabolic dysfunction or to be of microbial origin, with urease positive bacterial species implicated most often. Infectious calculi account for about 15-20% of all cases of urolithiasis and are often associated with biofilm colonization of a long-term indwelling urinary catheter or urethral stent. The use of closed catheter systems is helpful in reducing such problems; nevertheless, such a system only delays the inevitable, with infections emerging a little later. Various coatings intended to prevent the bacterial adhesion to the surface of catheters and implants and thus also the emergence of biofilm infections, unfortunately, do not inhibit the microbial adhesion completely and permanently and the only reliable method for biofilm eradication remains the removal of the foreign body from the patient.

Capillary isoelectric focusing of native and inactivated microorganisms.

The research of microorganisms includes the development of methods for the inactivation of viruses and other microbes. It also means to efficiently eliminate the infectivity of microorganisms without damage of their integrity and structure. According to the results of the last 5 years the capillary electromigration techniques appear to be very perspective for the comparison of the methods applicable for inactivation in the diagnostics and study of the pathogens. In this paper we suggest the capillary isoelectric focusing of the model microorganisms, Escherichia coli, Staphylococcus epidermidis, Candida albicans and bacteriophage PhiX 174, native or inactivated by different procedures. UV detection and fluorometric detection for the dynamically modified microbes by pyrenebutanoate on the basis of the non-ionogenic tenside were used here. Isoelectric points of native and/or dynamically modified microorganisms and other properties were compared with those obtained after microorganisms inactivation. The segmental injection of the sample pulse enabled the reproducible and efficient capillary isoelectric focusing in different pH gradients. The low-molecular-weight pI markers were used for tracing of the pH gradient.

Importance of biofilm in Candida parapsilosis and evaluation of its susceptibility to antifungal agents by colorimetric method.

The ability of C. parapsilosis (an important cause of nosocomial infections) to produce biofilm was evaluated in 32 bloodstream isolates and 85 strains isolated from skin. The biofilm formation was found in 19 (59%) blood isolates and only in 33 (39%) isolates from skin. The antifungal susceptibility was assessed for amphotericin B, itraconazole and voriconazole in planktonic and biofilm form of the 19 biofilm-positive bloodstream strains by broth microdilution method according to NCCLS standards. The method was modified by the use of resazurin as a colorimetric indicator of the metabolically active cells which makes the determination of the effect of antifungal agents easier. Biofilm forms of all strains were more resistant than their planktonic form.

[Methods for detection of biofilm formation in routine microbiological practice]. / Moznosti prukazu tvorby biofilmu v rutinní mikrobiologické praxi.

The increasing use of catheters, artificial implants and antimicrobials as well as high numbers of immunocompromised patients are major causes for concern over biofilm infections. These infections are characterized particularly by high resistance to antimicrobials and formation of persistent foci that may complicate therapy. Therefore, detection of biofilm formation is of high relevance to the clinician and his/her approach to the treatment. Reliable and sensitive methods for detection of this pathogenicity factor in clinically important organisms, suitable for use in routine microbiological laboratories, are needed for this purpose. Currently, a wide array of techniques are available for detection of this virulence factor, such as biofilm visualization by microscopy, culture detection, detection of particular components, detection of physical and chemical differences between biofilm-positive organisms and their planktonic forms and detection of genes responsible for biofilm formation. Since each of these methods has limitations, the best results can be achieved by combining different approaches.

Nuclear thyroid hormone receptors, alpha-glycerophosphate dehydrogenases, and malic enzyme in N-nitrosomethylurea-induced rat mammary tumors.

Specific L-3,3'5-triiodothyronine (triiodothyronine) binding by isolated nuclei was determined in N-nitrosomethylurea-induced mammary carcinomas and livers from intact and thyroidectomized animals. Tumors contained a single class of high-affinity nuclear triiodothyronine binding sites with characteristics similar to those of hepatic nuclei. Competition experiments showed the relative affinities of thyroid hormone structural analogues for both tumor and liver receptors to be: triiodothyroacetic acid greater than triiodothyronine greater than thyroxine greater than reverse triiodothyronine. Diiodotyrosine did not complete for the binding sites. Mammary tumors exhibited a wide range of binding sites, but most were in the range of 50 to 150 fmol/micrograms DNA. The levels were not affected significantly by hypothyroidism. Liver triiodothyronine receptor concentrations were approximately 5-fold those of tumors; they were unaffected by low serum thyroid hormone levels and were similar to those of non-tumor-bearing, euthyroid controls. Mitochondrial alpha-glycerophosphate dehydrogenase and cytosolic malic enzyme activities were reduced in both tumors and livers of hypothyroid rats; cytosolic alpha-glycerophosphate dehydrogenase levels were unchanged. Hepatic enzyme activities were similar in euthyroid tumor-bearing animals and in euthyroid healthy controls.

Variation in the binding of 125I-labeled interferon-beta ser to cellular receptors during growth of human renal and bladder carcinoma cells in vitro.

Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for 125I-labeled human interferon Beta ser IFN-beta ser). This recombinant produced interferon labeled with approximately one atom of 125I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000-2000/cell) with an affinity constant of 10(10)-10(11) L/M was measured at 4 degrees C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-beta ser. Major fluctuations in the binding of 125I-labeled IFN-beta ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P less than 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of 125I-labeled IFN-beta ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G0-G1. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of 125I-labeled IFN-beta ser 4-16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand. After an initial 40% decline in receptors per cell following serum stimulation, receptor concentration remained essentially unchanged. Induction of 2',5'-oligoadenylate synthetase in ACHN cells and antiproliferative activity in RT112, RT4, T24 (human transitional cell carcinoma), and ACHN cells by IFN-beta ser decreased significantly 48 h following plating. These changes in the biological activity of this interferon corresponded to growth related fluctuations in the IFN-beta ser binding.

Modulation of interferon receptor expression during combination beta ser-interferon and gamma-interferon treatment of human colon carcinoma cells.

Combination treatment of SKCO1 human colon carcinoma cells with beta ser-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma) results in a synergistic antiproliferative effect. The role of IFN-beta ser and IFN-gamma receptor modulation was investigated as a possible mechanism for this response. IFN-gamma (0.05-50 ng/ml) pretreatment of SKCO1 cells for 24 h decreased specific binding of 125I-IFN-beta ser by 35-60%. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-gamma showed that this reduction in binding was due to a decreased receptor affinity (control cells, Kd = 46 +/- 1.6 pM; IFN-gamma-treated cells, Kd = 106 +/- 6 pM, n = 2) rather than a significant change in receptor number (receptor number/control cell = 1214 +/- 471, receptor number/IFN-gamma treated cell = 1118 +/- 153, n = 2). In contrast, pretreatment of SKCO1 cells with IFN-beta ser (5 ng/ml) resulted in slight (10-35%) increases in 125I-IFN-gamma-specific binding. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-beta ser showed a decrease in binding affinity (control cells, Kd = 28 +/- 7 pM; IFN-beta ser-treated cells, Kd = 38 +/- 7 pM, n = 2) and a 32% increase in IFN-gamma receptor sites (receptor number/control cell = 4257 +/- 464, receptor number/IFN-beta ser-treated cell = 5570 +/- 730; n = 2). 125I-IFN-gamma internalization studies performed at 37 degrees C confirmed the cell surface binding assays; IFN-beta ser-treated cells internalized 30-50% more labeled IFN-gamma than untreated cells. However, it is unlikely that differences in binding and internalization of this magnitude play a primary role in the synergistic antiproliferative effect of IFN-gamma with IFN-beta ser in SKCO1 cells. Biochemical modulation at sites distal to the ligand receptor interaction should be investigated.

The influence of thyroidal status on rat hepatic mitochondrial NADH duroquinone reductase.

Hepatic mitochondrial NADH duroquinone reductase and alpha-glycerophosphate dehydrogenase activities were measured in rats with altered thyroidal status. Whereas alpha-glycerophosphate dehydrogenase activity was decreased in hypothyroid rats, NADH duroquinone reductase was increased approximately 3-fold in both thiouracil-fed and thyroidectomized rats. In hyperthyroid animals, NADH duroquinone reductase activity was decreased, whereas there was the expected elevation in mitochondrial alpha-glycerophosphate dehydrogenase. Maximum velocity measurements of NADH duroquinone reductase demonstrated that the increase in enzyme activity associated with hypothyroidism occurred without an alteration in Michaelis constants for the reaction. Rats bearing mammary carcinomas induced by N-nitrosomethylurea also showed an increase in hepatic NADH duroquinone reductase when rendered hypothyroid, but the enzyme was unaffected by thyroxine administration.

PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia.

OBJECTIVE: To assess the usefulness of polymerase chain reaction (PCR) assays in the diagnosis of fungal infections in immunocompromised patients. METHODS: A rapid and sensitive PCR-based assay for the detection and identification of fungal pathogens was designed and applicability of this method was investigated in a group of children with cancer and febrile neutropenia (FN). RESULTS: The ITS2 sequences and adjacent regions of 40 fungal pathogens were analyzed and primers for detection of all analyzed fungal species were designed. Amplification product length polymorphism (APLP) and restriction fragment length polymorphism (RFLP) generated genus- or species-specific patterns. The sensitivity of the method was approximately three cells of Candida albicans per 1 mL of blood. The results were available within 8 h after sample collection. The method was tested on 53 blood samples and one lung biopsy sample from 24 children with cancer and febrile neutropenia (FN). The PCR assay detected fungal DNA in 25 clinical samples from ten patients. Blood cultures were positive in only five samples, while another two blood-culture negative patients had positive cultures from throat swabs. The remaining 14 patients were both culture- and PCR-negative. Culture-isolated strains matched completely those obtained by PCR-APLP-RFLP identification. The identity of fungal species was confirmed by direct sequencing of amplified products. CONCLUSION: Our results suggest that PCR-APLP-RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.

Impact of surface coating on the adherence of slime producing and nonproducing Staphylococcus epidermidis.

The ability of Staphylococcus epidermidis to grow in the form of a biofilm not only facilitates its persistence in the host, but also allows it to survive at antibiotic concentrations several orders higher than the Minimum Inhibitory Concentration (MIC). We evaluated different surface treatments of hardened polystyrene in order to develop a model system for growth of S. epidermidis as a biofilm. We assayed for biofilm growth of S. epidermidis clinical isolates on unmodified polystyrene, on polystyrene modified by chemical abrasion and on polystyrene modified by sulfonation, using either Tryptic Soya Broth or Brain Heart Infusion as a growth medium. We concluded that sulfonated polystyrene and Brain Heart Infusion provided the best growth system for predicting the ability of a clinical isolate to form biofilm (Akaike value 23.680). Using this method, biofilm formation was detected in 14 (70%) of ica-positive strains and negative in 16 (80%) of ica-negative strains.

Biofilm detection and the clinical significance of Staphylococcus epidermidis isolates.

The ability of Staphylococcus epidermidis to produce biofilm was compared in 147 clinically significant strains repeatedly isolated from blood cultures of patients with bloodstream infection and in 147 strains isolated from skin. The strains were examined for the presence of ica operone, for the ability to form biofilm by Christensen's test-tube method and for the production of slime by Congo Red agar method. The ica operone was found in 92 (62.6 %) blood isolates and in 44 (29.9) isolates from skin. Christensen's test-tube method was positive in 79 (53.7) and 33 (22.4), Congo Red agar method in 64 (43.5) and 31 (21.1) of blood and skin isolates, respectively. All three methods were more frequently positive in clinically significant isolates from blood than in strains isolated from skin. The detection of ica operone and the Christensen's test-tube method showed better correlation with the clinical significance than the Congo Red agar method.