Initial attachment of Borrelia burgdorferi spirochetes to Vero cells.

BACKGROUND: Adhesion is the initial process in the establishment of any infection and can contribute to bacterial pathogenesis. Without the ability to adhere to host cell surface, there is no invasion, dissemination, or persistence and host colonization by many bacterial pathogens, including B. burgdorferi. During the infection, B. burgdorferi cells interact with cells of various origins. We are having limited information and knowledge regarding the borrelial invasion, intracellular existence and the host cell damage and the pathological effects to the host. We have investigated by electron microscope the adherence of motile Borrelia burgdorferi s.l. to Vero cells derived from the kidney of an African green monkey by electronmicroscopy. These cells have been shown as an interesting model for study of the toxic potential of many bacterial pathogens. METHODS: Adherence of the long-term in vitro passaged Borrelia burgdorferi sensu lato strains to a 24-hour monolayer of primate kidney epithelial Vero cells was studied using transmission electronmicroscopy. The reaction was read after incubation at 1-hour intervals. RESULTS: A vertical contact between borreliae and Vero cells was confirmed already after one hour of in vitro incubation. A cytotoxic effect of borreliae could be observed when the time of incubation was extended to 4 hour. The extent of attachment varied between the two borrelia strains tested. CONCLUSION: The optimal time for spirochetal adhesion in our model was 1 h postinoculation. Our results suggest that borrelia attaches to the tested cells by length and by the tip. The data showed that the extent of attachment varied between the two borrelia strains tested (Tab. 1, Fig. 4, Ref. 21).

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of Clostridium difficile isolates from three Czech hospitals in Eastern Bohemia in 2011-2012.

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

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).

Phenotypic characterization and putative virulence factors of human, animal and environmental isolates of Plesiomonas shigelloides.

Plesiomonas shigelloides (a bacterium widely distributed in aquatic ecosystems causing both intestinal and extra-intestinal diseases) shows a host of putative virulence markers, such as hemolysins, cytotoxins, production of exoenzymes associated with pathogenicity, adhesive ability and vacuolation of cell lines in vitro. Technical difficulties in detecting some of these virulence factors together with scantiness of epidemiological information, due to the lack of routine analysis for P. shigelloides as etiological agent of gastroenteritis, lead to sporadic and occasional finding of these bacteria. All this casts doubt on the real virulence potential of P. shigelloides and fuels a debate about assignment of these bacteria to the list of human pathogens. Here we demonstrated the phenotypic diversity and the putative virulence markers by examining serotype biochemical and virulence properties of 60 strains of P. shigelloides isolated from human, animal and environmental samples in different countries, which showed the unpredictable occurrence of the above properties depending on various locations and diverse sources.

[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.

Inhibition of mammalian cathepsins by Plesiomonas shigelloides.

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.

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.

Combined infection of Ixodes ricinus with three Borrelia burgdorferi sensu lato genotypes.

Ixodes ricinus ticks were collected by random collections from western and central Slovakia during the years 1996-98. The midgut content of 240 ticks was examined by dark-field microscopy and submitted for cultivation for the presence of borrelias. Spirochetes were found in 21 unfed and host-seeking adults and nymphs (8.8%). By the analysis of restriction fragment length polymorphism (RFLP) one sample from unfed I. ricinus male from western Slovakia was identified as a triple infection of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii. The simultaneous presence of different B. burgdorferi genospecies in one midgut sample (triple infection in the tick) could be observed only after the multipart amplification of denaturated DNA and subsequent pooling of the products for further analysis.

[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.

Immunochemical analysis of lipopolysaccharide-like component extracted from Borrelia burgdorferi sensu lato.

Immunoelectrophoresis and its modifications were applied to analysis of a lipopolysaccharide-like component (LPS-LC) extracted from Borrelia garinii strains K24 and K48 isolated from Ixodes ricinus and Borrelia burgdorferi sensu stricto strain B31. A modification of the hot phenol-water method was used for isolation of LPS. Immunoelectrophoresis (IE) and crossed immunoelectrophoresis (CIE) of LPS-LC with polyclonal rabbit antisera revealed a pattern and properties partially similar to LPS from other Gram-negative bacteria. B. garinii LPS-LC formed in CIE a diffuse band extending from the start to the anode. Similarly, the shape and position of the band in IE did not show major differences from LPS of other Gram-negative bacteria. The LPS-LC extracted from the three genomic groups of B. burgdorferi sensu lato were found to have similar immunochemical properties irrespective of their genotype origin.

[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.

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.

Specific detection of Plesiomonas shigelloides isolated from aquatic environments, animals and human diarrhoeal cases by PCR based on 23S rRNA gene.

Twenty-five strains of Plesiomonas shigelloides isolated from aquatic environment, 10 strains from human cases of diarrhoea and five strains from animals were identified by the polymerase chain reaction technique based on 23S rRNA gene. For this purpose, two primers targeted against part of the 5' half of the 23S rRNA gene of P. shigelloides (Escherichia coli number C-912, G-1195; Plesiomonas number C-906, G-1189) were designed. Results from our study indicated that this method might serve as a tool for a rapid and sensitive identification of P. shigelloides from different environmental and clinical sources.

[Immunostimulatory and other beneficial health effects of lactic acid bacteria]. / Imunostimulacné a iné zdravotne prospesné úcinky baktérií mliecneho kysnutia.

Lactic acid bacteria in functional foods can transiently colonize the intestine of man and exert beneficial probiotic effects. These were observed in a group of adult subjects administered daily by a lyophilized Enterococcus faecium M-74 in the form of waffles (Dr. Ebi) during nine weeks of a double blind placebo controlled clinical trial. The results showed significant immunostimulatory effect on both phagocytosis by neutrophils and antibody production.

Isolation, biochemical and serological characterisation of Plesiomonas shigelloides from freshwater in Northern Europe.

Isolation and characterisation of Plesiomonas shigelloides from fresh water in Northern Europe is reported in this study. The organisms were isolated from two lakes and a river in Sweden. All isolates of P. shigelloides showed an identical biochemical profile and belonged to different serotypes, namely, O18, O23, O26, O58 and O60. The study indicates that P. shigelloides may occur in the aquatic environment of cold climates and as a result, it is likely to be associated with human infections caused by waterborne pathogens in geographical areas with similar climatic conditions.