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).

[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.

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.

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.

Role of plasmids in virulence-associated attributes and in O-antigen expression in Shigella dysenteriae type 1 strains.

The association of plasmids with virulence characters and O-antigen expression was studied in two virulent and seven avirulent mutant strains of Shigella dysenteriae type 1. Deletion of a 12-Mda segment from a 140-Mda plasmid in two smooth avirulent mutants made the derivatives avirulent in the Sereny test and non-invasive in HeLa cells. The mutants were unable to bind Congo red, and did not express the virulence marker antigen. Mutants completely lacking the 140-Mda plasmid also showed similar avirulent characters. However, rough mutants retained the ability to bind Congo red. Our results indicate that the essential gene(s) for virulence may be located on the 140-Mda plasmid, a small deletion from which may lead to avirulence. This deletion did not affect the protein antigen expression nor change their antigenicity. Analysis of lipopolysaccharide (LPS) patterns showed that strains containing the 6-Mda plasmid produced the complete LPS and were smooth, whereas strains containing either the 140- or a 4- or 2-Mda plasmid, in the absence of the 6-Mda plasmid, produced smaller amounts of O antigen and were rough. Western-blot analysis and crossed immuno-electrophoresis gave similar results. The 140-, 4-, or 2-Mda plasmid, in the absence of the 6-Mda plasmid, may code for non-specific galactosyl transferase-like activity which can add, non-specifically and at a reduced level, the galactose residue (the first sugar in the O antigen repeat unit) to the LPS core.(ABSTRACT TRUNCATED AT 250 WORDS)

The association of haemagglutination and adhesion with lipopolysaccharide of Shigella dysenteriae serotype 1.

In this study the ability of strains of Shigella dysenteriae serotype 1 to agglutinate mammalian erythrocytes is attributed to the polysaccharide fraction of bacterial-cell lipopolysaccharide (LPS). LPS obtained from a rough, mutant strain of S. dysenteriae serotype 1, lacking the O-antigen polysaccharide side-chain, did not agglutinate erythrocytes, clearly demonstrating a link between O-antigen polysaccharides and haemagglutinating activity (HA). Strains of S. dysenteriae serotype 1 adhered well to cultured Henle Intestinal 407 cells, whereas rough strains adhered poorly. Pre-treatment of bacteria with LPS-specific antisera inhibited both HA and binding to cultured human-intestinal cells. The contribution of the polysaccharide side-chain and its associated HA--which appear to facilitate binding to cultured cells--to bacterial attachment to colonocytes and to the pathogenesis of shigellosis in vivo needs to be confirmed in animal studies.

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.

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.

International Training and Research in Central and Eastern Europe: The University of Iowa model.

In 1995, with support from the Fogarty International Center of the National Institutes of Health, The Center for International Rural and Environmental Health (CIREH) at The University of Iowa began developing the multi-level International Training and Research Program in Occupational and Environmental Health, focusing on countries in Central and Eastern Europe that were formerly under socialist control and have particular occupational and environmental health needs after decades of neglect and mismanagement. The purpose of the program is to prepare health science professionals to return to their home countries with new skills, added confidence, and leadership capabilities in public health to lead their colleagues and institutions in meeting the needs in their respective countries and establish active national and international networks and collaborations. By December 1998, 19 trainees had completed the five-month program and returned home to conduct workshops for others in their countries. Details of the program are presented.

Application of biotinylated and 32P probes for detection of P-fimbriae in urinary E. coli.

Escherichia coli is the common causative agent of urinary tract infections. Twenty-six strains of Escherichia coli were isolated from children with pyelonephritis, symptomatic urinary tract infections and asymptomatic bacteriuria. Biotinylated and 32P-DNA probes were prepared for detection of P-fimbriae in the isolates. Of the 13 strains isolated from patients with pyelonephritis 11 were positive for the presence of the P gene by both probes. Strains isolated from cases of symptomatic urinary tract infections revealed the presence of P gene only in three samples of the total of nine isolated. None of the isolated E. coli strains from asymptomatic bacteriuria was found positive for the presence of the P gene. The biotinylated probe was simple and easily applicable in standard laboratory conditions and therefore the authors recommend it for use in diagnostic laboratories.

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.

Occurrence of genes for P and S fimbriae and hemolysin in urinary Escherichia coli.

Escherichia coli is the common causative agent of urinary tract infections. Sixty-one strains of E. coli isolated from children with urinary tract infections were tested by colony hybridization for the presence of genes determining P and S fimbriae and hemolysin. Of these strains, 46 possess a gene for hemolysin, 44 for P fimbriae and 28 for S fimbriae. Only 30 strains formed lytic zones around the colonies on plates with sheep erythrocytes. The results indicated that simultaneous occurrence of genes in urinary E. coli was highest for P fimbriae and hemolysin and lower for other combinations of the tested genes.

Adherence of intestinal and extraintestinal Pseudomonas aeruginosa to tissue culture cells.

The adherence pattern of Pseudomonas aeruginosa strains to HeLa, Vero and CHO cells was studied. The diffuse type of adherence was found to prevail on HeLa cells. It was characteristic for intestinal and environmental strains. Urinary strains revealed more often a localized adherence. A similar pattern was obtained with CHO cells. Experiments with Vero cells showed an equal distribution of intestinal strains regarding the diffuse, localized and mixed adherence. Urinary strains revealed mostly a localized adherence of a similar pattern as was observed on HeLa and CHO cells.

Immunochemical properties of Vibrio cholerae LPS.

Immunochemical analysis of LPS isolated from Vibrio cholerae O1 and non O1 showed that this macromolecular complex shares common antigenic epitopes in the sugar moiety. The epitopes can be detected after mild alkaline hydrolysis of LPS in vitro. Membrane-associating activity of both O1 and non O1 LPS did not indicate any differences of the species.

Spirochetal non-Borrelia-microorganism isolated from Ixodes ricinus.

Spirochetal microorganisms were isolated from female Ixodes ricinus in Slovakia. Morphological, immunochemical and molecular biological analysis showed that the microorganism shared several common antigens with Borrelia species while other genetic traits were distinct and not related to Borrelia burgdorferi sensu lato. Lyme disease patient's serum contained antibodies reacting with antigens of this microorganism. On the one hand the cross-reacting antigens represent a risk of false positive results in laboratory diagnostics, while on the other hand they have a certain potential for vaccine development against Lyme disease.

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.

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.