Effect of aminoglycosides on surface hydrophobicity of Acinetobacter baumannii

Effects of amikacin, gentamicin, netilmicin and tobramycin at subinhibitory concentrations (sub-MICs) (11/4, 1/8, 1/16 or 1/32 of their MICs) on the cell surface hydrophobicity of two Acinetobacter baumannii strains (7194 and 16265) were evaluated. Hydrophobicity was determined by two different methods - by adherence of bacteria to hydrocarbon (xylene) and by aggregation of bacteria in ammonium sulphate solutions at various concentrations. The adherence of A. baumannii strains to xylene decreased, mainly, after treatment with netilmicin at 1/4, 1/8 or 1/16 of the MIC (to 6.4%, 17.0% or 24.5% of the control value) (strain 7194) and after treatment with amikacin and gentamicin at 1/4 of their MICs (to 58.4% or 54.4%) (strain 16265). A decrease in surface hydrophobicity of exposed strains under these conditions was shown in salting-out test, too. Tobramycin reduced hydrophobic properties of A. baumannii strains at all tested sub-MICs to only a small extent.     

Cell Surface Hydrophobicity and Slime Production of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> Brazilian Isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M − <2M, SAT 2M − <4M, and SAT ≥4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT ≥ 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0–0.4 O.D. group, including non-glass adherent isolates; 0.5–0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8–1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT. Received: 13 May 2002 / Accepted: 5 July 2002     

Toxinogenicity and markers of pathogenicity of Pseudomonas aeruginosa strains isolated from patients with tumor diseases

Potential virulence factors (elastase, proteinase, lipase, phospholipase C, alginate) as well as surface properties (hydrophobicity, motility) were determined in 103Pseudomonas aeruginosa strains isolated from patients with cancer. Nontypable strains were the dominant group (60%), followed by serotypes O11 (17%), O12 (7%) and O4 (5%). Seventy-one strains (69%) produced high level of elastase (10–60 mg/L), 87% of the strains possessed high activity of proteinase (bacterial) (10–250 mg/L) and 69% of the strains demonstrated higher level of lipase (20–150 U/mL); these elevated levels of enzymes were associated mainly with nontypable strains. On the other hand, 79% of the strains did not produce or produced only a low level of phospholipase C and 60% of isolates did not manifest any or very low production of alginate. Hydrophobicity demonstrated by adherence of the bacteria to xylene was shown by 69% of strains; 94% of strains aggregated with ammonium sulfate. Motility in the range of 31–80 mm was found in 76 strains (74%). The considerable virulence of testedP. aeruginosa strains was confirmed. The nontypable strains manifested the most frequent group with high level of elastase, proteinase, lipase, hydrophobicity and motility.     

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Abstract Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction ( P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.     

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity.     

Susceptibility of Brazilian Staphylococcal Strains to Glycopeptides Evaluated by Different Testing Methods

Reports of staphylococci with reduced susceptibility to glycopeptides are cause for concern. This study evaluated the susceptibility of 84 staphylococci clinical isolates to glycopeptides by the disk diffusion, agar dilution, E-test, and BHIA screening methods. Vancomycin agar dilution showed all strains presented minimum inhibitory concentration (MIC) ranging from 0.5 to 2 μg/ml, and the E-test showed similar results. Teicoplanin agar dilution test showed MICs ranging from ≤ 0.5 to 2 μg/ml for Staphylococcus aureus and MICs ranging from <0.25 to 32 μg/ml for coagulase-negative Staphylococcus (CNS). Ten CNS isolates presented MICs ranging from 8 to 32 μg/ml for agar dilution and/or E-test. All the staphylococci were susceptible to vancomycin by the disk diffusion test (DDT), but two CNS isolates presented intermediate resistance to teicoplanin by the DDT and MICs of susceptibility, with two other CNS strains, teicoplanin-susceptible by the DDT, presented MICs of intermediate resistance. On the vancomycin-containing agar, 20 CNS isolates were able to grow, but no S. aureus strain. All these isolates showed MICs to teicoplanin (4–32 μg/ml) higher than those isolates that did not grow on the agar screen plate. PFGE of chromosomal SmaI digests showed a wide diversity of these CNS strains, without any predominance of a single PFGE pattern. Received: 25 May 2001 / Accepted: 9 July 2001     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Pukyongia salina gen. nov., sp. nov., a novel genus in the family Flavobacteriaceae

A Gram-negative aerobic bacterium, designated RR4-38T, was isolated from a biofilter in a seawater recirculating aqua-culture system (RAS) in Busan, South Korea. The bacteria were irregular, short, rod-shaped, non-motile, oxidase-positive, and catalase-negative. Growth of the strain RR4-38T was observed at 15–35·C (optimum, 25–30·C), pH 5.5–9.5 (optimum, pH 8.0), and in the presence of 0–5% (w/v) NaCl (optimum, 3%). Phylogenetic analysis based on the 16S rRNA gene sequences showed that the strain RR4-38T formed a distinct lineage with close genera Ulvibacter (≤ 95.01% 16S rRNA gene sequence similarity), Aureitalea (94.74%), Aureisphaera (≤ 93.27%), and Jejudonia (93.07%) that all belong to the family Flavobacteriaceae. Whole-genome sequence comparison revealed that the ANI (average nucleotide identity) and digital DDH (DNA-DNA hybridization) values between strain RR4-38T and the two closest strains, Ulvibacter antarcticus DSM 23424T and Aureitalea marina S1-66T, were 68.96–69.88% and 17.4–19%, respectively. The genome analysis revealed that the strain might be involved in biodegradation of organic debris produced by farmed fish in aquaculture systems. The predominant respiratory quinone was menaquinone MK-6 and the major cellular fatty acids were iso-C15:0 (26.5%), iso-C17:0 3-OH (16.4%), iso-C15:1 G (15%), and iso-C16:0 3-OH (9.6%). The major cellular polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, unidentified aminolipids, and glycolipids. Based on phenotypic, chemotaxonomic, and phylogenetic features, strain RR4-38t represents a novel genus and species in the family Flavobacteriaceae, for which the name Pukyongia salina gen. nov., sp. nov. is proposed. The type strain is RR4-38T (= KCTC 52651T = DSM 108068T).     

Pharmacodynamic parameters and hydrophobicity changes induced by meropenem in Acinetobacter baumannii.

The suppression of bacterial growth of four Acinetobacter baumannii strains after 60 min exposure to meropenem at supra-inhibitory concentrations (postantibiotic effect; PAE) or at supra-subinhibitory concentrations (postantibiotic-sub-MIC effect; PA SME) was studied. The duration of the PAE was dependent on antibiotic concentration and on the strain. Meropenem at 2x or 4x the minimum inhibitory concentration (MIC), with the exception of one strain treated with 4x MIC, did not provoke suppression of bacterial growth compared with untreated controls. The highest concentration of meropenem (8x MIC) induced PAE for the strains tested in the range of 0.6-6.9 h. The effect of supra-subinhibitory concentrations of meropenem (2x, 4x or 8x MIC + 0.2x MIC) on bacterial growth was more efficient compared with supra-inhibitory concentrations alone. Two out of the four strains treated did not renew their growth. Bacterial suspensions exposed to meropenem showed reduced surface hydrophobicity. Decreases in hydrophobicity were associated with longer PAE and PA SME depending on the strain.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

ClinicalPseudomonas aeruginosa: Potential factors of pathogenicity and resistance to antimicrobials

Resistance to 17 antimicrobials, surface hydrophobicity, motility, biofilm, production ofN-acylhomoserine lactone signal molecules (N-butyrylhomoserine lactone andN-3-oxolauroylhomoserine lactone) and response to oxidative stress were analyzed in 47 clinicalPseudomonas aeruginosa strains. In addition to natural resistance, the strains demonstrated the greatest level of resistance to cefotaxime (91.5 %). Isolates in the range of 44.7–57.4 % were resistant to aminoglycosides and ciprofloxacin, of 25.5–36.2 % to cephalosporins. On the other hand, 97.9 % remained susceptible to meropenem, 93.6 % to piperacillin + tazobactam and 87.2% to piperacillin. The majority of the strains (72.3 %) manifested their hydrophilic character. Higher zones of motility showed 12 isolates (in average 54.8 mm) as compared to the others (30.2 mm). Approximately 1/3 of the strains (29.8 %) produced a higher amount of biofilm quantified by measuring the absorbance of solubilized crystal violet (0.20–0.46) than the rest of isolates (0–0.19). All but two strains producedN-3-oxolauroylhomoserine lactone and in 48.9 % of samplesN-butyrylhomoserine lactone were detected. Only four isolates with higher biofilm production showed both types of homoserine lactone. Majority of the strains (70.2 %) manifested higher resistance to H₂O₂ than the rest of the strains. The group of strains resistant to aminoglycosides and ciprofloxacin revealed a significantly higher number of hydrophobic strains (compared with the sensitive ones). In contrast, higher number of strains sensitive to aminoglycosides and ciprofloxacin or only to ciprofloxacin producedN-butyrylhomoserine lactone and biofilm (compared to the resistant ones). Such association was not found among the rest of the tested parameters. The results indicate that the resistance to antimicrobials inP. aeruginosa isolates was not generally associated with changes in the production of the pathogenicity factors.     

Antimicrobial susceptibility, β-lactamase and enterotoxin production in <Emphasis Type="Italic">Bacillus cereus</Emphasis> isolates from clinical and food samples

The antimicrobial susceptibility of 30 clinical and 30 food Bacillus cereus isolates was determined. All isolates were susceptible to streptomycin, ciprofloxacin and gentamicin, 90 % of them to clindamycin and vancomycin, and 67 % to erythromycin. All isolates were resistant to amoxicillin with clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, cloxacillin, cefotaxime with clavulanic acid and penicillin. The MIC values (determined by E-tests) were 48–256 mg/L for ampicillin, 0.19–1.5 mg/L for gentamicin, 0.125–1.0 mg/L for clindamycin, 0.047–4.0 mg/L for erythromycin and 1.5–16 mg/L for vancomycin. The MICs 4.6–18.75 g/L were observed for penicillin using the microdilution method. The presence of metallo-β-lactamases was detected by E-test for 100 % of strains. Nonhemolytic diarrheal enterotoxin (NHE) was produced by 98.3 % of strains, while 31.7 % of them produced hemolytic diarrheal enterotoxin (HBL). Clinical isolates produced 10 % more HBL than food isolates. The psychrotrophic strains isolated from food samples produced NHE at 6.5 °C in 73 % of cases.     

Preliminary assays for the development of a probiotic for goats

In order to determine probiotic properties, 137 strains of lactic acid bacteria from the feces of Creole goats were screened, only six were resistant to pH 2.0 and bile salts (0.3%). Three strains identified as Lactobacillus and two as Enterococcus showed agglutination with the treated yeast. Between them, Lactobacillus DDL17, DDL19, DDL48 and Enterococcus DDE39 demonstrated high specificity in this test because the correspondent agglutination was inhibited by one sugar, suggesting the presence of a lectin-like structure in their cell walls, which could be due to adhesion ability. Another Enterococcus strain (DDE55) showed low affinity because five sugars inhibited the agglutination of the treated yeasts. The results of hydrophobic properties showed that the strains who were able to agglutinate yeasts presented similar hydrophobic characteristics as hexadecane, xylene and toluene, but high specificity was not related to a high hydrophobicity. Only two strains (Lactobacillus DDL19 and DDL48) showed aggregation with the lowest concentration of ammonium sulfate, complementing the hydrophobicity assay. Only one strain, Lactobacillus DDL48, showed an inhibition against an enteric indicator strain (Salmonella Typhimurium and Escherichia coli O111). This inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. These strains could be pre-selected in order to complete studies focused on designing a probiotic for use in goat feed.     

Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.     

<Emphasis Type="Italic">Singulispaera mucilagenosa</Emphasis> sp. nov., a novel acid-tolerant representative of the order <Emphasis Type="Italic">Planctomycetales</Emphasis>

Two novel strains of budding bacteria, Z-0071^T and Z-0072, were isolated from dystrophic humified waters formed by xylotrophic fungi in the course of spruce wood degradation. The cells of both strains are coccoid (0.95–1.80 μm), nonmotile, single or arranged in pairs. The cells have a complex system of intracellular membranes and are covered with fimbriae and surrounded by a mucous capsule up to 0.3 μm thick. Both strains are aerobic organoheterotrophic, mesophilic, and acid-tolerant microorganisms that are able to grow under microaerobic conditions. They utilize N-acetyl-glucosamine, carbohydrates, and lactate as growth substrates. The strains grow in a pH range of 4.0–7.5 with an optimum at 6.0–6.5. The temperature range for growth is 4–30°C, with an optimum at 25–28°C. Strains Z-0071^T and Z-0072, inhabitants of dystrophic low-mineral waters, are NaCl-sensitive: the NaCl content in the media above 0.5 g/l inhibited growth. The main fatty acids of strains Z-0071^T and Z-0072 are C_16:0, C_18:1ω9c, and C_{18:2ω9c, 12c}. The DNA G + C base content is 51.2–51.7 mol %. The sequences of the 16S rRNA gene fragments (1310 bp) of strains Z-0071^T and Z-0072 were found to be identical. The obtained sequences showed a 94.3% similarity with the sequences of the type strain of the most closely related species Singulisphaera acidiphila MOB10≅T. The phenotypic and phylogenetic properties of strains Z-0071^T and Z-0072 support classification of these strains within the genus Singulisphaera as a new species Singulisphaera mucilagenosa sp. nov., with the type strain Z-0071^T (VKM B-2626).     

Halorussus rarus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern

Two halophilic archaeal strains TBN4^T and TBN5 were isolated from Taibei marine solar saltern in Jiangsu, China. Both strains showed light red-pigmented colonies and their cells were rod, motile and Gram-stain-negative. They were able to grow at 25–50°C (optimum 37°C), at 1.4–4.3 M NaCl (optimum 2.1 M NaCl), at 0–1.0 M MgCl₂ (optimum 0.005 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0). Their cells lyse in distilled water and minimal NaCl concentration to prevent cell lysis is 8% (w/v). The major polar lipids of the two strains were PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester), PGS (phosphatidylglycerol sulfate) and five glycolipids chromatographically identical to S-TGD-1 (sulfated galactosyl mannosyl glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), TGD-1 (galactosyl mannosyl glucosyl diether), DGD-1 (mannosyl glucosyl diether) and DGD-2 (an unknown diglycosyl diether). Phylogenetic analysis revealed that TBN4^T and strain TBN5 formed a distinct clade with genus Haladaptatus (showing 90.0–90.9% 16S rRNA gene similarities). The DNA G + C content of strain TBN4^T and strain TBN5 are 66.1 and 65.4 mol%, respectively. The DNA–DNA hybridization value between strain TBN4^T and strain TBN5 was 94.3%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain TBN4^T and strain TBN5 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Halorussus rarus gen. nov., sp. nov. is proposed. The type strain is TBN4^T (=CGMCC 1.10122^T = JCM 16429^T).     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia. Received: 4 April 1997 / Accepted: 10 June 1997     

In Vitro Activities of Fourteen Antimicrobial Agents Against Drug Susceptible and Resistant Clinical Isolates of Mycobacterium tuberculosis and Comparative Intracellular Activities Against the Virulent H37Rv Strain in Human Macrophages

Minimal inhibitory concentrations (MICs) of 14 first and second-line antituberculous drugs against drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis (including the multiple drug-resistant or MDR-TB isolates), as well as the type strain H37Rv, were determined radiometrically by the Bactec 460-TB methodols. MICs (μg/ml) of all the fourteen drugs were within an extremely narrow range in case of susceptible strains; isoniazid (0.02–0.04), rifampin (0.2–0.4), ethambutol and streptomycin (0.5–2.0), ethionamide (0.25–0.5), D-cycloserine (25–75), capreomycin (1–2), kanamycin (2–4), amikacin (0.5–1.0), clofazimine (0.1–0.4), ofloxacin (0.5–1.0), ciprofloxacin (0.25–1.0), and sparfloxacin (0.1–0.4). The activity of second-line drugs remained unaltered against MDR-TB isolates resistant to routine first-line drugs. With peak serum level concentrations (Cmax), the intracellular killing of the virulent H37Rv strain was studied in detail in cultured human macrophages. Based on an decreasing order of bactericidal activity, our results showed the following spectrum of intracellular drug action: among the first-line drugs, rifampin > ethionamide = isoniazid > ethambutol > streptomycin > D-cycloserine; among second-line drugs, clofazimine = amikacin > kanamycin = capreomycin; among fluoroquinolones, sparfloxacin > ofloxacin > ciprofloxacin. On the other hand, contrary to atypical mycobacteria, the macrolide drug clarithromycin was inactive against both extracellular and intracellular M. tuberculosis. Received: 23 January 1996 / Accepted: 5 April 1996     

Postantibiotic effects of gentamicin and netilmicin onSerratia marcescens: Effects on hydrophobicity and motility

The impact of postantibiotic effect (PAE) of aminoglycosides (gentamicin, netilmicin) on cell-surface hydrophobicity and motility of a clinical isolateSerratia marcescens was evaluated. For the induction of PAE 2× and 4×MIC concentrations of both antibiotics were used. Gentamicin and netilmicin induced a PAE of similar duration after 2×MIC concentration (2.7 and 2.8 h, respectively). Both aminoglycosides demonstrated concentration-dependent PAE. At a concentration of 4×MIC they produced PAEs of 5.9 and 8.2h, respectively. The evaluation of hydrophobic properties ofS. marcescens after affecting PAE showed that both aminoglycosides inhibited adherence to xylene. This inhibition was also concentration-dependent. More expressive, was netilmicin which inhibited the adhesion by 70.5% at 2×MIC and by 85.2% at 4×MIC. Netilmicin inhibited also the adhesion to nitrocellulose filter by 34.7% at 4×MIC. Exposure of the bacterial cells to suprainhibitory concentrations of both aminoglycosides resulted only in moderate inhibition of motility of strain tested compared to the unexposed cells.