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Candida dubliniensis is a recently described yeast that causes infections in mucosal surfaces as well as sterile body sites. Candida dubliniensis develops resistance to fluconazole (FLC) more rapidly than the closely related species C. albicans. The killing activity of amphotericin B (AMB), 5-fluorocytosine (5FC), FLC, voriconazole (VRC) and posaconazole (POS) was determined against six C. dubliniensis clinical isolates, identified using molecular biological methods and C. dubliniensis CD36 reference strain. Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute standard procedure. Time-kill assays were performed using RPMI-1640 as test media over a 48-h period. AMB proved to be fungicidal at >or=0.5 microg ml(-1) against all clinical isolates after 48 h. 5FC was only fungicidal at 32-64x MIC (4-8 microg ml(-1)) against all C. dubliniensis isolates. FLC, VRC and POS were fungistatic; decrease in colony number was observed only at the highest concentrations tested (8, 4 and 4 microg ml(-1), respectively). Triazoles invariably showed fungistatic effect at concentrations attainable in the serum. In clinical situations when a fungicidal antifungal is desirable, AMB may be used.

The STD Department of Semmelweis University Budapest is the National Centre of Hungary, which is responsible for screening and care of sexually transmitted diseases (STD), including syphilis and gonorrhoea. 42,114 patients attended the STD Department and 25,362 anonymous screening (HIV: 12,337, syphilis: 13,025) were done between January 2005 and December 2008. During this period 600 syphilitic and 339 gonorrhoea infections were diagnosed. The obligatory HIV screening of patients with sexually transmitted infections (STI) resulted positive result in 47 cases, and 63 patients infected with HIV acquired new syphilitic or gonorrhoea infection. Contact tracing was successful in around 400 syphilis cases, and 150-200 gonorrhoea cases per year. We present our statistical data in order to call attention to the resurgence of syphilis and gonorrhoea and the importance of STD co-infections.

Antimicrobial peptides are considered to be viable alternatives to conventional antibiotics. However, they rarely show systemic efficacy in animal models when added at non-toxic doses. The dimer A3-APO was designed to attack both the bacterial membrane and the Enterobacteriaceae-specific domain of the heat shock protein DnaK in order to reduce toxicity whilst maintaining activity. The peptide exhibited a minimal inhibitory concentration (MIC) range of 2-128 mg/L against 28 clinical Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium strains, with a median MIC of 30 mg/L. At this concentration, A3-APO was bactericidal to E. coli 5770, a fluoroquinolone-resistant extended-spectrum beta-lactamase-producing strain. The No Observed Adverse Effect Limit (NOAEL) at repeated intraperitoneal peptide administration was 20mg/kg. When administered at this dose three times starting immediately after E. coli Neumann infection, A3-APO cured 100% of mice in a standard bacteraemia model used by the pharmaceutical industry. In a more stringent assay, when treatment started after E. coli 5770 bacteraemia had already been established, three doses of 10mg/kg A3-APO prolonged early survival at a rate similar to that of imipenem and reduced the bacterial counts to base level. When the second assay was repeated in kidney clearance conditions resembling those in humans, 10mg/kg A3-APO was as efficacious as imipenem in the long-term. The increased in vivo efficacy compared with the in vitro bactericidal figures can potentially be explained by the generally observable immunostimulatory properties of antimicrobial peptides. Peptide A3-APO shows promising features as a member in our antibiotic arsenal against multidrug-resistant bacterial pathogens.

The purpose of this study was to characterise verotoxigenic Escherichia coli (VTEC) strains isolated in Hungary from 2000 to 2006. Altogether, 33 human VTEC strains were investigated to define the O:H antigens, verotoxin 1, 2 (vtx1 and 2), intimin (eae), enteroaggregative heat-stable toxin (ast1), autoagglutinating adhesin (saa) and enterohaemolysin (ehlyA) genes and sensitivity to 11 antimicrobial agents. The strains belonged to 14 different O:H serotypes, among which O157:NM (non-motile) was the most prevalent (45%, 15/33). Patients infected with O157 more often presented bloody diarrhoea or haemorrhagic colitis (63%, 12/19) than those infected with non-O157 (46%, 6/14). Haemolytic uraemic syndrome evolved in two patients infected with O26:H11. The vtx1vtx2c toxin gene combination was found in 58% (11/19) and vtx2c alone in 31% (6/19) of the O157 strains. All of the O157 strains possessed gamma1, while two O26 strains had the beta1 intimin gene. Twenty strains (75%, 25/33) carried the ehlyA gene and five non-O157 strains had ast1. The majority of the strains (76%) were resistant to at least one antimicrobial agent, but none of them showed the extended-spectrum beta-lactamase (ESBL) phenotype.

Killing activity of amphotericin B, fluconazole, voriconazole, posaconazole, and 5-fluorocytosine was determined against 6 Candida parapsilosis , 3 Candida orthopsilosis , and 4 Candida metapsilosis clinical isolates. After 24 h, 1 of 6 C. parapsilosis , 1 of 3 C. orthopsilosis , and 3 of 4 C. metapsilosis isolates were killed at 1 to 4 μg/mL (1–8× MIC) amphotericin B. The remaining isolates were killed by 2 to 4 μg/mL amphotericin B after 48 h. Fluconazole was fungistatic at ≥1× MIC (0.5–2 μg/mL) against C. parapsilosis and at ≥2× MIC (4–8 μg/mL) against C. orthopsilosis and C. metapsilosis isolates. Voriconazole inhibited C. parapsilosis at ≥1× MIC (0.015–0.12 μg/mL), but the other 2 species were inhibited only at 4 to 8× MIC (0.25–0.5 μg/mL). Against C. orthopsilosis and C. metapsilosis , posaconazole was fungistatic close to the MIC (0.03–0.06 and 0.015–0.03 μg/mL, respectively). Against C. orthopsilosis and C. metapsilosis , fluconazole and voriconazole, but not posaconazole, seem to be less active in vitro than against C. parapsilosis .

Fifteen nosocomial cases of extended-spectrum β-lactamase-producing Klebsiella pneumoniae occurred among 132 neonates in a premature intensive care unit in Hungary in June through November 1998. Fourteen strains were indistinguishable by molecular biological typing and harbored the same single conjugative extended-spectrum β-lactamase-encoding plasmid that was spontaneously found in a Serratia marcescens strain in the same patient.

Udgivelsesdato: 2008-Mar Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.

Summary The opsonic requirement for phagocytosis and killing and cell-surface hydrophobicity of five strains of Staphylococcus saprophyticus isolated from clinical sources were studied. Phagocytosis and killing of bacteria by human granulocytes were measured in suspension. Bacterial aggregating cell-surface hydrophobicity was determined by salt aggregation, and the absorptive hydrophobicity was measured by hydrophobic interaction chromatography. All strains were well opsonised by pooled normal human serum 10%. Ingestion of these bacteria could be detected to a variable extent in the absence of extracellular opsonins; heat-inactivated serum 10% or intravenous IgG concentrate 1 mg/ml improved phagocytosis of all strains. Significantly increased rates of both the ingestion and killing of one of the five strains occurred in the presence of IgG or in the absence of opsonins, compared to those found with each of the other four. This particular strain had significantly stronger adsorptive surface hydrophobicity than the other four strains, and with all strains there was a correlation between hydrophobicity and phagocytosis by granulocytes in the absence of opsonins.

The water-soluble N-methoxy-PEG-yl-, N-beta-D-glucopyranosyl- and N-beta-D-maltosylthioureido aglyco-ristocetin were prepared which, in contrast to ristocetin A, did not induce thrombocyte aggregation. The antibacterial activity of N-beta-D-maltosylthioureido aglyco-ristocetin A against MRSA was comparable to that of ristocetin A, while its activity against Enterococcus faecalis (VRE, TSE) is somewhat stronger when compared to those of vancomycin and ristocetin A.

The incidence of fluoroquinolone resistance among Hungarian routine laboratory Streptococcus pneumoniae isolates, collected in 2000-2002, in common with other European countries, was very low; only 5/304 strains (1.64%) were resistant to ciprofloxacin (MIC = 4 microg/ml), and the other fluoroquinolones showed full efficacy. However, we could identify the Lys-137-Asp amino acid change, caused by a point mutation in the QRDR of the parC gene, in five strains. Additionally, we observed a definite shift in the minimum inhibitory concentrations (MICs) of all fluoroquinolones towards higher values throughout the study period. These two findings, coupled with the increasing consumption figures of fluoroquinolones, suggest that pneumococcal resistance looks poised to develop in Hungary.

The aglycones of the antibiotics eremomycin, vancomycin and ristocetin (3, 4 and 6, respectively) were prepared by deglycosidation of the parent antibiotics with hydrogen fluoride, and complete assignation of their 1H, 13C and 15N spectra was performed. The squaric acid amide esters (11-14), were prepared from dimethyl squarate. The corresponding asymmetric diamides (16-19, 22, 23) were also synthesized using 4-phenylbenzylamine and triglycine. The advantage of the method is the high regioselectivity and that no protecting group strategy is required. Electrospray mass spectroscopic method was elaborated for the determination of the site of substitution of the modified antibiotics. The antibacterial activity of the prepared compounds is discussed in detail.

The authors review the novel data available in the literature and their experimental results about the enterotoxins of Staphylococcus aureus. Their genetics, phylogenetics, biological characteristics, as well as mechanisms of action are dealt with. Furthermore, pathogenesis, pathomechanism, immunology, major symptoms and signs, diagnostics, management, possible outcomes, and epidemiology of the Staphylococcus aureus enterotoxin-associated human diseases are hereby reviewed.

In Hungary more than 25% of Staphylococcus aureus strains and 33.4-70.1% of strains of different coagulase-negative Staphylococcus species isolated from inpatients in the last five years were methicillin-resistant. These are great clinical problems because the methicillin-resistant strains are resistant to all beta-lactam antibiotics, furthermore, most of them exhibit multiple resistance, too. For these reasons, the fast and exact microbiological diagnosis is of utmost basic and life-saving importance. In this review the most important evidences for proving pathogenicity and virulence of methicillin-resistant S. aureus strains and our knowledge on the origin, genetic background, conditions of expression, forms of appearance, phenotypical and genotypical properties of methicillin resistance are summarised. Phenotypical semiquantitative and quantitative methods aiming the detection of methicillin resistance are shown with their advantages and restrictions. Genetic methods applicable to detect mecA gene responsible for coding methicillin resistance, furthermore, those by which clonal and genomic relationship and spread can be proven in epidemic cases are presented. Finally, up-to-date schemes for a proper identification as well as systemic and local elimination of methicillin resistant Staphylococcus strains are suggested.

Eukaryotic tissue culture appears to be a suitable model for measuring the bacterial cytotoxic effect. Propionibacterium acnes strains were isolated from corneal tissue removed by keratoplastic surgery from patients with corneal dystrophy or bullous keratopathy. The cytotoxic effect of the filtrates of 10 P. acnes strains were studied by means of measuring the decrease of the mitochondrial dehydrogenase activities of viable epithelial (HeLa) and fibroblastic (BHK-21) cell cultures. A time and concentration dependent, reversible cytotoxic effect was detected in both tissue types. The results also showed that strains of P. acnes are capable of surviving anaerobic conditions for as long as 8 months and suggest that production cytotoxic effects during the long persistence it may harm human tissue.

Due to the revolutional development in sampling devices, transport media, automated equipments, microvolume probes and molecular microbiological techniques in the last decade new possibilities have become available for diagnosing microbiologically congenital infections in both the mother and the foetus and the newborn. This minireview gives an insight into the diagnostic tuls of clinical microbiology and infection serology by showing laboratory diagnostic processes of most frequent protozoal, viral and bacterial infections step-by-step. It exhibit an almost complete list of transplacental, intrauterinal and connatal infections. Attention is also focused to the complexity of the interrelationship between the clinical microbiology and infection serology data concerning the infections of the mother and her offspring.

The effects of the alkali metals sodium, potassium and lithium on the growth and on certain virulence factors (adhesion, cell-surface hydrophobicity and the germinating ability) of Candida albicans were investigated. It can be concluded that high concentrations of alkali metals possessed an inhibitory effect on the growth of the Candida cells and preincubation in the presence of alkali metals had a negative effect on all the virulence factors studied. It is worth emphasizing that the changes induced during the preincubation persisted even when the high concentrations of the alkali metals were removed from the cell suspension. However, even at high concentrations of sodium or potassium a considerable growth of Candida cells could be measured. Data also showed that although alkali metals could significantly decrease certain virulence traits of the fungus they could not totally inhibit either the adhesion or the germ tube formation potential of the cells. Thus, in spite of the high salt concentrations Candida cells may represent a health hazard in such habitats.

A new system, Micronaut- Candida , was compared to API ID32C to identify 264 yeast ( Candida albicans , C. parapsilosis , C. tropicalis , C. krusei , C. inconspicua , C. norvegensis , C. lusitaniae , C. guilliermondii , C. dubliniensis , C. pulcherrima , C. famata , C. rugosa , C. glabrata , C. kefyr , C. lipolytica , C. catenulata , C. neoformans , Geotrichum and Trichosporon species, Rhodotorula glutinis , and Saccharomyces cerevisiae ) clinical isolates. Results were in concordance in 244 cases. Eighteen out of the 20 of discordant results were correctly identified by Micronaut- Candida but not by API ID32C, as confirmed by PCR ribotyping.

L-[4,5-3H]isoleucine was introduced to label anteiso-fatty acid (AIFA)-containing lipids in Staphylococcus aureus SG 511. After an overnight incubation in peptone broth in the presence of 37 kBq L-[4,5-3H]isoleucine/ml, 8.5-13% of the total radioactivity applied was found to be incorporated into the cells. 22.4-25.6% of the incorporated radioactivity was found in AIFA-containing lipids extracted by chloroform-methanol-water (2:1:0.2, v/v/v) at pH 2. The interphase contained 70-75% of the incorporated radioactivity. Lipoteichoic acid, extracted by phenol-water (80:20, w/v) contained less than 1% of the incorporated radioactivity, as measured after purification by hydrophobic interaction chromatography on octyl sepharose gel. Within 1 h after addition of 10 micrograms/ml penicillin G to exponentially growing cultures of S. aureus, that led to non-lytic death of the cells, 11.9-18.1% of the incorporated L-[4,5-3H]isoleucine label were released. Lipids containing AIFA were excreted to 5.4-8.4% of total incorporated activity; this amount represents more than 1/4 of the labeled cellular lipids.

The methodology to determine bacterial cell-surface hydrophobicity is discussed as well as the possible contribution of various surface-bound bacterial components to the hydrophobic nature of a cell surface. A number of agents and conditions affect cell-surface hydrophobicity, such as growth media, growth-phases and bacterial species. Studies are presented on hydrophobicities of staphylococcal cells of different species in connection with slime production and growth in serum-soft agar. Experimental data support a correlation between hydrophobicity and phagocytosis. The genetic origin of hydrophobicity, and the therapeutical implication of hydrophobic interactions are also discussed.

In this report we examined the glycopeptide susceptibility of enterococci, isolated in 2005, from slaughtered animals, within the confines of Hungarian Antibiotic Resistance Monitoring System. We determined the presence of the van genes as well as their genetic relatedness in enterococci from poultry. Enterococcus sp. strains (n = 175) were collected from intestinal samples of slaughtered poultry in 2005. The origin of the samples was registered at county level. After screening the strains with 30 mg vancomycin disc 19 (86%) intermediate resistant and 4 (3%) fully resistant strains were found. The distribution of minimum inhibitory concentration (MIC)-values among 23 enterococcus strains which were intermediate or resistant to vancomycin were 0.25 mg/L (4.4%), 2 mg/L (8.6%), 4 mg/L (8.6%), 8 mg/L (61%), 16 mg/L (8.6%) and 256 mg/L (8.6%). The MICs of teicoplanin were 0.25 mg/L (4.3%), 1 (8.6%), 4 mg/L (78.3%), 16 mg/L (4.3%) and 256 mg/L (4.3%). The two most vancomycin-resistant strains were vanA carriers (1 E. faecalis and 1 E. faecium ).The farms that produced these strains can be reservoirs of VRE and the affected farms should change the technology of disinfection and breeding in order to prevent the emergence of high numbers of human VRE isolates in Hungary.

The surface hydrophobicity of cells of Staphylococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohydrate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydrophobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and aggregating properties did not produce surface capsule or slime. Heat treatment (60° C or 80° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating properties.

An improved salt aggregation test (improved SAT) was developed to sensitize the determination of bacterial cell-surface hydrophobicity. One drop of a fresh bacterial suspension standardized to an A1cm540 of 20 (equivalent to 5 × 109 cfu/ml), and one drop each of ammonium sulphate solutions stained with methylene blue, were mixed on a white hydrophobic paper card using toothpicks. The bacterial suspensions, methylene blue stock solutions and the ammonium sulphate solutions (0.01–4.0 M) were made in 0.02 M sodium phosphate buffer, pH 6.8. Bacterial aggregations were read immediately after mixing the salt/bacterial suspensions while the card was gently rocked. Readings were also confirmed the next day on dried preparations. The results proved independent of reading time and mixture conditions (wet or dry preparations). The improved SAT technique is very rapid and sensitive, the reaction is easily read with the naked eye, and the paper cards can be stored for documentation of aggregation patterns after drying. In the improved SAT, the Staphylococcus cells of different species aggregated in 5 ways: tiny, medial, flaky granular, particulated and macrofilamentous forms; Salmonella strains aggregated in flaky granular, particulated and macrofilamentous forms.

The bacteriostatic and bactericidal activities of cefetamet (Ro 15-8074) were compared with those of cefadroxil, cefaclor, ampicillin, doxycycline, chloramphenicol and erythromycin against a total of 400 bacterial strains isolated from nosocomial respiratory tract and auditory canal infections. The broth macrodilution method was used to determine antimicrobial susceptibility. None of the compounds was effective against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus strains. Cefetamet was very active against ampicillin-resistant Klebsiella pneumoniae. Proteus mirabilis and Escherichia coli strains with the minimal inhibitory concentrations for 90% of the strains tested ranging from 0.25 to 1 mg/l. Cefetamet at a concentration of 8 mg/l inhibited 90% of Enterobacter aerogenes and 60% of S. epidermidis strains. In biophotometric studies, subinhibitory concentrations of cefetamet greatly lengthened the lag-phase and flattened the log-phase, the extent varying with the concentrations and species tested. It did not cause detectable lysis in the stationary-phase of the cultures. Adding two to 32 times inhibitory concentrations of cefetamet to middle-log cultures, no or moderate lysis occurred., except with Esch. coli. This observation suggests that cefetamet is primarily bacteriostatic for some of the strains tested.

Methicillin resistance in a highly resistant mutant of Staphylococcus aureus could not be diminished by elevated temperature. After 100 subcultures at 43°C the resistance to methicillin was retained when determined at 37°C.