Temperature and pH affect the production of bacterial biofilm.

The effect of different cultivation temperatures (30 and 37 degrees C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4-98.4 %) in the biofilm production was observed at 37 degrees C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 degrees C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103-143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139-244 %, at pH 7.5 136-164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151-319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113-177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204-329 % at pH 8.5, and 123-316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).

[Aminoglycosides and colistin inhibit biofilm formation in Klebsiella pneumoniae]. / Aminoglykozidy a kolistin potlácajú tvorbu biofilmu u Klebsiella pneumoniae.

OBJECTIVE: To evaluate the effect of subinhibitory concentrations (sub-MICs) of amikacin, tobramycin and colistin on biofilm formation, surface hydrophobicity, lipase activity and response to oxidative stress in two clinical K. pneumoniae strains. METHODS: Biofilm formation was quantitatively determined by a crystal violet absorption assay, surface hydrophobicity was measured by adherence of bacteria to xylene, lipase activity was determined by the spectrophotometric method with Tween-20 as a substrate and oxidative stress was visualized as a zone of clearing around the disc soaked with hydrogen peroxide. RESULTS: The antibiotics significantly reduced bacterial biofilm formation in a dose-dependent manner. They were most effective at concentrations of 1/2 and 1/4 MIC. Biofilm formation was inhibited by 1/2 MICs of amikacin to 21.2% (strain 39) and 22.6% (61/P), of tobramycin to 25.1% (39) and 19.5% (61/P) and of colistin to 7.4% (39) and 19.1% (61/P) of the control values (no antibiotic). Similarly, 1/4 MICs reduced biofilm formation to 28.6% (39) and 28.9% (61/P) of the control levels for amikacin, to 35.3% (39) and 20.5% (61/P) for tobramycin and to 8.7% (39) and 20.4% (61/P) for colistin. Cultivation of the strains with the antibiotics at 1/16 MICs was least effective in inhibiting biofilm formation. It was reduced to 80.4% (39) and 97.7% (61/P) of the control levels for amikacin, to 69.4% (39) and 64.4% (61/P) for tobramycin and to 61.3% (39) and 74.7% (61/P) for colistin. The tested strains were strongly hydrophilic and changes in surface hydrophobicity caused by antibiotics were negligible. Most antibiotic treated strains showed mildly increased sensitivity to oxidative stress and decreased lipase activity (with the exception of colistin in strain 39). CONCLUSION: Amikacin, tobramycin and colistin at sub-MICs considerably reduced biofilm formation K. pneumoniae strains, in most mildly increased sensitivity to oxidative stress, decreased lipase activity but practically did not affect adherence to xylene.

[Biofilm formation and response to oxidative stress in Pseudomonas aeruginosa and Vibrio cholerae non-O1 depending on culture media]. / Tvorba biofilmu a odpoved' na oxidacný stres u kmenov Pseudomonas aeruginosa a Vibrio cholerae non-O1 v závislosti na kultivacných médiách.

OBJECTIVE: To evaluate the effect of six culture media (five complex and one mineral) on biofilm formation and response to oxidative stress in Pseudomonas aeruginosa (3 strains) and Vibrio cholerae non-O1 (3 strains). METHODS: Biofilm formation was quantitatively determined by a crystal violet absorption assay. The bacterial response to oxidative stress evoked by hydrogen peroxide was visualized as a zone of clearing around the disc after 24 h incubation at 37 degrees C. RESULTS: For both of the bacterial species studied, biofilm formation was the highest after cultivation in tryptone soya broth (TSM) or in TSM supplemented with 8% glucose (TSM+GL), being the lowest in mineral medium (MM). V. cholerae non O1 strains were 1.4 to 4.3 times more responsive on average to oxidative stress depending on culture medium as compared with P. aeruginosa strains. The culture medium had no significant effect on H2O2 evoked by response to oxidative stress in vibrios in contrast to P. aeruginosa. In P. aeruginosa, the highest mean resistance to H2O2 was observed after cultivation in peptone water while the most sensitive cells were found after incubation in TSM+GL and MM. CONCLUSION: The culture medium composition influnced biofilm formation in both of the bacterial species tested and had a considerable effect on response to oxidative stress in P. aeruginosa.

Immunological properties of complex conjugates based on Vibrio cholerae O1 Ogawa lipopolysaccharide antigen.

Host protection by humoral immunity against Vibrio cholerae O1 confers lipopolysaccharide (LPS)-specific vibriocidal antibodies. Levels of relevant specific antibodies are closely related to complement-mediated inactivation of the vibrios inoculum, especially on the mucosal surface of intestine. We have tested complex V. cholerae O1 Ogawa-detoxified lipopolysaccharide (dLPS) conjugates. The first conjugate contained glucan both as the immunomodulator and the matrix; the second conjugate contained immunologically inert amylose as matrix. Both d-LPS conjugates contain multiply attached dLPS antigen. These conjugates elicited a statistically significant increase of antigen-specific IgG levels in mice (P<0.001 and P<0.05, respectively). The specific anti-conjugate IgG and IgA response after the second (booster) dose were significantly higher compared to pre-immune and whole-cell response. The most effective vibriocidal activity was observed in the case of conjugate, with glucan as the matrix. The highest correlation was found between vibriocidal activity and specific IgG2b (r=0.765) and IgA (r=0.887) sera levels. The determination of specific IgG subclasses and IgG2a + 2b/IgG1 ratio revealed a dominant T(H)1 cell response crucial for effective vaccine candidate.

Clinical pseudomonas aeruginosa: potential factors of pathogenicity and resistance to antimicrobials.

Resistance to 17 antimicrobials, surface hydrophobicity, motility, biofilm, production of N-acylhomoserine lactone signal molecules (N-butyrylhomoserine lactone and N-3-oxolauroylhomoserine lactone) and response to oxidative stress were analyzed in 47 clinical Pseudomonas 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 produced N-3-oxolauroylhomoserine lactone and in 48.9% of samples N-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 produced N-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 in P. aeruginosa isolates was not generally associated with changes in the production of the pathogenicity factors.

[Biological characteristics of Plesiomonas shigelloides strains of human and animal origin]. / Biologické vlastnosti kmenov Plesiomonas shigelloides humánneho a veterinárneho pôvodu.

We focused on serotyping and biological characteristics of Plesiomonas shigelloides strains potentially associated with virulence. Thirteen strains isolated from humans (H) and 14 strains of animal origin (A) were tested. The most frequent serotype among H strains was 040:H6 while 066:H3 predominated among A strains. All of the H strains and 92.8% of A strains were hydrophobic. H isolates showed lower motility (30.1 mm) compared to A isolates (46.8 mm). As many as 76.9% and 71.4% of H and A strains, respectively, produced beta-hemolysis. Both H and A strains exhibited low biofilm production on a glass surface. No significant differences were found between H and A strains in lipase production and histidine decarboxylase production. The zones of bacterial growth inhibition in response to oxidative stress were on average 26.6 mm and 28.1 mm for H and A strains, respectively. None of the strains tested produced unsubstituted short-chain acyl homoserine lactones. Our results showed that tested Plesiomonas shigelloides strains produced multiple potential virulence factors that may play a role in the pathogenesis of infections caused by this agent.

[The role of iron in bacterial virulence]. / Uloha zeleza v bakteriálnej virulencii.

Data on the role of iron in host-bacterium interaction in relation to virulence are summarized. Attention is focused on host iron acquisition pathways in bacteria. Host iron can be acquired by several mechanisms, e.g. from hemoglobin degradation products such as heme and hemin, directly from ferrated transferrin and lactoferrin, indirectly from iron binding proteins by the production of siderophores and from intracellular iron stores (ferritin). Regulation of iron uptake is discussed.

[Type III bacterial secretion systems and their relation to virulence]. / Bakteriálne sekrecné systémy typu III a ich vzt'ah k virulencii.

Newer data on type III secretion systems (SST systems) associated with virulence in gramnegative bacteria are summarized. The focus is on substrates of SST systems (effector proteins), recognition of type III secretion signals, structural components of SST systems and their genetic determination.

Potential virulence-associated properties of Plesiomonas shigelloides strains.

Serotyping and some potential virulence-associated markers were investigated in Plesiomonas shigelloides strains isolated from humans, animals and aquatic environments. Surface properties of these strains were evaluated using Congo red binding, salt-aggregation test, bacterial adherence to xylene and motility. Production of pancreatic elastase, proteinase (consistent with subtilisin Carlsberg), triacylglycerol lipase, histidine decarboxylase and beta-hemolysin was also determined. In addition, detection of signal molecules such as C4-C8 unsubstituted N-acylhomoserine lactones (AHLs) was performed. The serological typing of the P. shigelloides strains showed that the isolates belonged to 13 different serovars. The majority of the strains were hydrophobic and motile. The strains produced low levels of elastase, proteinase and histidine decarboxylase whereas triacylglycerol lipase activity was relatively high. Only 23.3 % of the strains produced hemolysin. The AHLs signal molecules were not detected. P. shigelloides strains were able to produce a variety of potential virulence markers which may be involved in the pathogenesis of Plesiomonas-associated infections.

[Bacterial cellular communication in relation to pathogenicity]. / Bakteriálna bunková komunikácia vo vzt'ahu k patogenite.

The authors briefly summarize data on cell-cell communication (quorum sensing) as well as principles of simple methods for detection of N-acyl homoserine lactones.

The effect of disinfectants on Plesiomonas shigelloides.

The effects of ten commercially available disinfectants on virulence associated properties of Plesiomonas shigelloides were tested. All the disinfectants tested contained quaternary ammonium salts. The majority of the disinfectants when used at subinhibitory concentrations increased surface hydrophobicity as evaluated by bacterial adherence to xylene and decreased bacterial motility in a concentration dependent manner. Disinfectants did not significantly affect lipase activity. However, more than half of the antimicrobials tested increased the resistance of bacteria to hydrogen peroxide. The disinfectants, in a similar manner to antibiotics at concentrations below MIC, interfered with potential virulence factors of Plesiomonas shigelloides.

Influence of growth media on potential virulence factors of Pseudomonas aeruginosa.

Potential virulence factors of three Pseudomonas aeruginosa strains after growth in three complex media (CM) and in one mineral medium (MM) were evaluated. Cell surface hydrophobicity demonstrated by adherence of bacteria to xylene as well as enzymatic activity (elastase, protease, lipase) of the strains grown in CM varied with composition of CM and with strain. All strains cultivated in CM showed higher hydrophobicity and higher elastase, protease and lipase (with the exception of one strain) activity in comparison with bacteria incubated in MM. Even no production of elastase was detected in the strains after growth in MM. Motility of bacteria was affected by culture media the least. In vitro composition of growth media influenced some potential virulence factors of P. aeruginosa.

[Effect of tobramycin on characteristics of strains of Acinetobacter species]. / Vplyv tobramycínu na niektoré charakteristiky kmenov Acinetobacter species.

Suppression of bacterial growth in seven strains of the Acinetobacter species after 30 min treatment with tobramycin at suprainhibitory concentrations (postantibiotic effect--PAE) and at supra-sub-inhibitory concentrations (postantibiotic effect of subinhibitory concentrations--PA SME) as well as changes in surface hydrophobicity and in the production of lipase and histamine in the exposed strains were studied. Pharmacodynamic parameters (PAE and PA SME) as well as changes in bacterial characteristics tested were dependent on antibiotic concentration and on the strain used. Suppression of bacterial growth after treatment with tobramycin at 2 x MIC was in a range of 0.6-4.5 h, a higher concentration (4 x MIC) induced a longer PAE (1.9-5.4 h). Tobramycin at supra-subinhibitory concentrations (2 x MIC + 0.2 x MIC and 4 x MIC + 0.2 x MIC) caused total suppression of bacterial growth. In the majority of the tobramycin-treated strains, an increase in hydrophobicity manifested by adherence of bacteria to xylene as well as an increase in lipolytic activity was observed. Tobramycin at the concentrations tested did not affect the production of histamine.

[Characteristics of strains of Acinetobacter spp. after culture in various media]. / Niektoré vlastonosti kmenov Acinetobacter spp. po kultivácii v rôznych médiách.

Growth, surface properties, production of lipase, histamine and some signal molecules (N-acyl-homoserine lactones, AHLs) in four Acinetobacter baumannii strains, in one Acinetobacter lwoffii and in one Acinetobacter haemolyticus strain cultivated in three complex media were studied. More intensive growth of bacterial suspensions was observed in Mueller-Hinton broth and in brain heart infusion in comparison with growth in proteose peptone. Greater growth of bacterial strains (with the exception of A. haemolyticus) in these two media was associated with more significant surface hydrophobicity of bacteria manifested by their adherence to xylene. A higher level of lipase was found in all strains (with the exception of A. haemolyticus) after cultivation in Mueller-Hinton broth. Composition of culture medium did not affect production of histamine. None of the studied strains in any medium tested produced short-chained unsubstituted AHLs that can be ones of the signal molecules shared with cell-to-cell communication.

Some properties of Plesiomonas shigelloides treated with aminoglycosides.

The effect of aminoglycoside antibiotics (amikacin, gentamicin, netilmicin and tobramycin) at sublethal concentrations (sub-MICs) on some properties of Plesiomonas shigelloides strains was evaluated. All agents decreased the bacterial surface hydrophobicity. Amikacin (1/4 of the MIC) and netilmicin (1/4 and 1/8 of the MIC) changed the hydrophobic character of P. shigelloides surface to a hydrophilic one. Treatment of the strains with aminoglycosides decreased also motility, netilmicin being the most effective. No significant changes were found in lipolytic activity of antibiotic-treated strains. In the majority of cases aminoglycosides increased sensitivity of bacteria to hydrogen peroxide. The tested antibiotics did not induce production of short-chained N-acylhomoserine lactones signal molecules. Aminoglycosides at sub-MICs affected important activities of P. shigelloides potentially associated with their virulence in dependence on strain, antibiotic and concentration.

Characteristics of clinical Acinetobacter spp. strains.

Resistance to 13 antimicrobial agents, resistance to the bactericidal activity of human serum, hydrophobic properties, lipolytic activity and production of histamine were determined in a total of 50 clinical Acinetobacter spp. strains (A. baumannii, A. lwoffii, A. calcoaceticus, A. haemolyticus). None of the tested isolates showed resistance to meropenem and none of A. lwoffii, A. calcoaceticus and A. haemolyticus strains were resistant to amikacin. Forty-six strains (92%) manifested resistance to ampicillin, 90% to cefuroxime, 68% to ciprofloxacin, 58% to piperacillin, gentamicin and cotrimaxazole, 50% to cefotaxime, 44% to amikacin, 42% to ceftazidime, 38% to piperacillin/tazobactam, 24% to netilmicin and 16% to ampicillin/sulbactam. In particular, A. baumannii and A. calcoaceticus strains showed considerable antibiotic resistance. Thirty-one isolates (62%) showed serum resistance; intermediate sensitivity was found in 19 isolates (38%). The majority of the strains (72%) demonstrated a strongly hydrophobic character; 16% of isolates exhibited moderate hydrophobic properties. All strains showed lipolytic activity; production of histamine was detected in 14 of 43 strains examined.

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 103 Pseudomonas 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 tested P. aeruginosa strains was confirmed. The nontypable strains manifested the most frequent group with high level of elastase, proteinase, lipase, hydrophobicity and motility.

[Effect of culture media on surface and enzymatic activity in Klebsiella species]. / Vplyv kultivacného média na povrchové a enzymatické aktivity Klebsiella species.

The effect of three complex media (KM) [Mueller-Hinton broth (MHB), brain heart infusion (MSI) and proteose peptone (PP)] and one mineral medium (MM) on surface and enzyme activities of five strains Klebsiella species was studied. Klebsiella oxytoca and Klebsiella ornithinolytica had a marked hydrophobic character after growth in MHB, MSI and MM, Klebsiella terrigena only in PP. K. oxytoca and K. ornithinolytica had a higher motility after cultivation in KM compared with MM, the motility of K. terrigena was not affected. The lipolytic activity of all tested strains was highest after growth in MSI and PP. The composition of culture medium affected bacterial parameters tested to a different extent depending on the species.

In vitro effect of ampicillin/sulbactam on Acinetobacter species.

The effects of subinhibitory concentrations (sub-MIC) of ampicillin/sulbactam (AMP/sulbactam), on the surface hydrophobicity and the lipase activity of ten Acinetobacter strains, were examined. The alterations in the activities studied were strain and drug concentration dependent. Most of the strains treated showed a decrease in surface hydrophobicity to a different extent. The hydrophobic character of three strains exposed to 1/4 or 1/8 of the MIC of the antibiotic was changed to a hydrophilic state. The majority of Acinetobacter strains after treatment with antibiotic possessed increased lipolytic activity. AMP/sulbactam even at sub-MIC may interfere with possible virulence factors of Acinetobacter strains in vitro.

[Klebsiella species from the viewpoint of nosocomial infections and virulence factors]. / Klebsiella species z hl'adiska nozokomiálnych nákaz a faktorov virulencie.

The author reviewed data on Klebsiella species mainly in connection with their participation in nosocomial infections, with their antibiotic resistance and production of ESBL. Potential virulence factors of Klebsiella are also evaluated.