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Training future programming teachers requires an innovative approach. Not only students need to handle the most current trends in technologies and teaching-learning methodologies, but also they must develop the capacity and criteria to search and select the most adequate to their context. This work analyzes the application of a collaborative Research-Based Learning methodology in the Programming subject of a master's degree in teacher training. The objective was to create a digital learning ecosystem and analyze the impact on the development of programming teaching skills. The results show that students perceive positive effects on the development of teaching skills, generating useful resources. However, teamwork has conditioned the quality of such resources. The digital ecosystem has allowed students to share knowledge with their peers and forthcoming students. Students who already had the generated ecosystem available valued it very positively. Future programming teachers require lifelong learning which can be supported by this living ecosystem.

A study is presented to compare different methodologies for the cross-sectional visualization of membrane-electrode assemblies (MEAs) with SEM. Four techniques for cutting MEAs have been tested and analyzed: the sharp-edge cutting, CO2 laser, embedded-mechanical polishing, and ion-milling. Characteristics of each procedure are compared. Sharp-edge cutting, being fast and low cost, may preserve layers porous morphology rather unaltered, but obtaining a good cross-section in a wide area is stochastic and with low probability. CO2-laser beam cutting alters severely carbon and Nafion layers as a consequence of the burning of carbon based materials. Embedded-mechanical polishing is a well stablished and reproducible method that shows accurately the thickness of the layers, however it is too aggressive with highly porous structures that may appear smoothed or removed. Ion-milling is also good for accurate thicknesses measurements, and more conservative with the porous morphologies than the previous one, but they are also smoothed.

Mechanical properties of membrane-electrode assemblies (MEAs) for proton exchange membrane fuel cells (PEMFCs) have been studied with the small-punch technique. MEAs were operated according to a non-accelerated aging protocol, with changes in humidification and cell temperature. After operation, degradation of the MEA is reflected by a decrease in power density, increase in the internal resistance, and decrease in open circuit voltage. Small disks (8 mm diam.) were taken from different locations of the degraded MEA to test the mechanical properties by small-punch. The results show an increase in plastic deflection and a decrease in maximum load of degraded MEAs, that result in lower apparent Young modulus. The degradation is larger in the area close to oxygen outlet. SEM microscopy shows that degradation affects majorly to the fibers of the carbon cloth gas diffusion layer (GDL). Combined small-punch and SEM correlate the change in mechanical properties on aged MEA with carbon fibers degradation. Such GDL degradation must also be principal responsible for the increase in the internal resistance.

Development projects of different types mainly aim to alleviate poverty and ameliorate the livelihoods of local people. One of the strategies commonly used is to focus on organizations and build from their existing capacities in order to improve their living standards or try to build new organizations to work in a common project. Social and human capitals are two key components of these organizations and they might be crucial to the success of the actions that they accomplish. Both can be considered as part of the social capacity of the local organization. This capacity can be enforced with development projects through capacity building. This term means much more than training activities as it includes not only human resource development but also organizational and institutional development (UNESCO, 2010). Capacity and capacity building concepts, as well as capacity measurements in this context are explored to build a framework to evaluate the social capacity generated with the interventions and better plan the actions to be undertaken by the projects to succeed. The focus is set on rural development projects.

Plesiomonas shigelloidesis a Gramnegative pathogenic bacterium belonging to the Enterobacteriaceae family. To date, only few lipopolysaccharide LPS structures from P. shigelloidesstrains are known. In particular, three core oligosaccharides have been found. Recently, we elucidated the structure of the Oantigen of P. shigelloides30273 serotype O1 and in this paper we present the characterization of the core structure from the LPS of the same strain. The LPS was hydrolyzed under both alkaline and mildly acidic conditions. In both cases, a mixture of oligosaccharides was obtained, which was purified by gel filtration and HPAEC. The oligosaccharides were characterized by chemical analysis, 2D NMR spectroscopy and MALDITOF mass spectrometry. A new core structure was found for P. shigelloides. In particular, from the analysis of the acid hydrolysed product it was possible to reveal the presence of a of DglyceroDtalo2octulopyranosonic acid Ko residue, which substitutes in part the terminal 3deoxyDmannooct2ulosonic acid Kdo unit. The Ko residue is not frequently found in core structures.© WileyVCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009

Plesiomonas shigelloidesis a Gram-negative bacterium belonging to the Enterobacteriaceaefamily. It has been found in an aquatic environment in the tropical and subtropical regions and is responsible for many gastrointestinal infections in humans, which take place from drinking untreated water or eating uncooked shellfish. Plesiomonas shigelloideshas also been reported to provoke extraintestinal infections such as meningitis and bacteremia in immunocompromised adults and neonates. Despite the emerging importance of this pathogenic microorganism, only three different O-antigens have been characterised so far. The structure of the O-chain of the lipopolysaccharide (LPS) from Plesiomonasshigelloidesstrain 302–73 (serotype O1) was determined by chemical analysis, 1D and 2D NMR spectroscopy and MALDI-TOF mass spectrometry. The polysaccharide was constituted by a linear pentasaccharidic repeating unit as follows: →3)-α-L-PneNAc4OAc(1→4)-α-L-FucNAc(1→4)-α-L-FucNAc(1→4)-α-L-FucNAc(1→3)-β-D-QuiNAc4NHb(1→ (PneNAc = 2-acetamido-2,6-dideoxy-talose, Hb = (S)-3-hydroxybutanoyl) PneNAc O-acetylation was not stoichiometric and was found to be about 75 . The position of the O-acetyl group and the amount of acetylation were deduced by NMR spectroscopic analysis. All the monosaccharides included in the repeating unit were deoxyamino sugars, which most probably, together with the presence of O-acetyl groups, were responsible for the recovery of the LPS in the phenol layer of the phenol/water extract of dried bacteria cells.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

ABSTRACTThis work aimed to establish the role of gne(encoding UDP-GalNAc 4-epimerase activity) and galE(encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificusbiotype 2, serovar E. DNA sequence data revealed that gneand galEare quite homologous within this species (≥90% homology). Mutation in gneof strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galEdid not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gnewas correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificusbiotype 2.

This work aimed to establish the role of gne (encoding UDP-GalNAc 4-epimerase activity) and galE (encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificus biotype 2, serovar E. DNA sequence data revealed that gne and galE are quite homologous within this species (≥90% homology). Mutation in gne of strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galE did not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gne was correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificus biotype 2.

Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O– phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

ABSTRACTMesophilic Aeromonas hydrophilastrains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O−phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gnegene is present in all the mesophilic Aeromonasstrains tested. No changes were observed for the LPS core in a gnemutant from A. hydrophilastrain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophilaAH-3. Some of the pathogenic features of A. hydrophilaAH-3 gnemutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gnemutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonasstrains, possess two kinds of flagella, and the absence of O34 antigen molecules by gnemutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gnegene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O-:K- phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

ABSTRACTKlebsiella pneumoniaestrains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O−:K−phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The ugegene was present in all the K. pneumoniaestrains tested. The K. pneumoniae ugemutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single ugewild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented ugemutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

A transposon mutant from Aeromonas hydrophilaAH‐3 was obtained which was highly resistant to opsonophagocytosis. The mutation was identified in the ftsEgene and we characterised the operon ftsY, Eand Xfrom this bacterium. These genes, as in enteric bacteria, are neighbours to rpoH. The A. hydrophila ftsEand Xgenes were fully able to complement Escherichia coli ftsEmutants, and also complement the opsonophagocytosis‐resistant phenotype of the A. hydrophilamutant strain. This phenotype seems to be related to the filamentous phenotype at 37°C exhibited by the A. hydrophila ftsEmutant.

Aeromonas hydrophilaAH‐3 strains carrying mutations in mgtE, which encodes a Mg2+and Co2+transport system, showed a 50% reduction of in vitro adherence to HEp‐2 cells, a reduction in swarming in semisolid swarming agar, and decrease in biofilm formation of over 60% in comparison to the wild‐type strain. The cloned A. hydrophila mgtEexpressed from a plasmid complements a Salmonella typhimuriumstrain deleted for all Mg2+transporters both phenotypically and by measurement of 57Co2+uptake. Likewise, plasmid‐borne mgtEwas able to complement the changes observed in A. hydrophila mgtEmutants. We suggest that MgtE and thus Mg2+and possibly Co2+have a role in A. hydrophilarelated to their swarming ability and related consequences such as adherence and biofilm formation.

ABSTRACTThe adherence mechanism of Aeromonas caviaeSch3N to HEp-2 cells was initially investigated through four mini-Tn5mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes,flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmAand flmBgenes were present in all mesophilicAeromonasspp. (A. hydrophila, A. caviae, A. veroniibv. veronii, andA. veroniibv. sobria) tested, this was not the case for the neuA-flmD-neuBgenes. Construction and characterization of flmBinsertion mutants in five other mesophilic Aeromonasstrains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonasto human epithelial cells; (ii)flmAand flmBare genes widely distributed in the mesophilic Aeromonasand are involved in flagella assembly, and thus adherence; and (iii) in A. caviaeSch3N the flmAand flmBgenes are found in a putative operon together with neuA, flmD, andneuBand are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

ABSTRACTOne representative recombinant clone encoding Klebsiella pneumoniaeO5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library ofK. pneumoniaeKT769 (O5:K57) introduced intoEscherichia coliDH5α. A total of eight open reading frames (wbO5gene cluster) were necessary to produce K. pneumoniaeO5-antigen LPS in E. coliK-12. The enzymatic activities proposed for thewbO5gene cluster are in agreement with the activities proposed for the biosynthesis of K. pneumoniaeO5-antigen LPS. Using the complete DNA sequence of the K. pneumoniae wbO5gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5−mutants and the corresponding wild-type strains or complemented mutants with the wbO5gene cluster (O5+strains), we found that the presence of K. pneumoniaeO5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats.

ABSTRACTWe cloned and sequenced the structural gene for Aeromonas hydrophilaporin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that of ompFinEscherichia coli, is adjacent to aspCand transcribed in the same direction. However, upstream of the porin II gene no similarities with E. coliwere found. We obtained defined insertion mutants in porin II gene either in A. hydrophila(O:34) or A. veronii sobria(serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.

ABSTRACTTwo different representative recombinant clones encodingAeromonas hydrophilalipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophilaAH-3 (serogroup O34) introduced into Escherichia coliDH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants ofA. hydrophilaAH-3 defective in either plaorplcgenes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophilaAH-3 plcmutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plcsingle gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonassp. serogroup O:34 infection process.

ABSTRACTWe have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniaewhich overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniaeclinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+and half O-strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.

The complement resistance ofAeromonas salmonicidastrains grown under conditions promoting capsule formation was investigated using well characterized strains and their isogenic mutants. Complement resistance was previously studied using the same strains growing under noncapsulating conditions. The serum resistant strains were found to activate complement, but rapidly degrade C3b preventing productive formation of the lytic complex C5b-9. Isogenic lipopolysaccharide rough mutants grown under non-capsulating conditions were serum sensitive, binding a large amount of C3b and leading to productive formation of C5b-9. When grown under conditions promoting capsule formation, these mutants were partially resistant to complement because less C3b is bound to them and also partially degraded, with a concomitant reduction in lytic C5b-9.

The capsular polysaccharide (CPS) ofKlebsiella pneumoniaeis an important virulence factor. Salicylate, which inhibits CPS production, was used to expose subcapsular antigens and components that may play an important role in host defense. Salicylate treatment greatly increased phagocytosis of five O1 serotypes by human polymorphonuclear leukocytes with normal rabbit serum and rabbit antisera against purified O1 lipopolysaccharide (O1LPS) as opsonins (p<0.01 or <0.05). Similar results were obtained with rabbit antiserum against a non-encapsulated isogenic strain. To further determine how salicylate increases susceptibility to phagocytosis, the binding of monoclonal antibodies against O1LPS or the LPS core and the binding of complement component C3b were measured by ELISA. The data indicate that salicylate reduced the barrier of CPS in serotypes O1:K1, O1:K10, and O1:K16 and unmasked subcapsular antigenic components in serotypes O1:K2 and O1:K66 so that bound opsonins could react with receptors on phagocytes. Serum bactericidal assays supported this conclusion. Therefore, decapsulating agents such as salicylate accentuate phagocytosis ofK. pneumoniaeby making subcapsular antigens and components accessible to immune and nonimmune host defences and vaccination with subcapsular antigens may exhibit optimal protection against lethal infection when combined with salicylate therapy.

We compared the ability of Aeromonas hydrophilawild‐type strains of serogroup O:34, non‐motile Tn5aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophilaAH‐3 (O:34, wild‐type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophilaserogroup O:34 bacterial invasion of both fish cell lines.

We compared the ability of differentAeromonas hydrophilastrains from serogroup O:34 grown at different temperatures to colonizein vivothe germfree chicken gut. We found a good colonization when the strains were grown at 20°C but not when they were grown at 37°C. We previously described that these strains were able to form the O-antigen lipopolysaccharide (LPS) when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen LPS (rfbmutants), and showed that they were unable to colonize the germfree chicken gut. All these results prompted us to conclude that the O-antigen LPS, in these strains, is a main factor for colonization in this animal model system.

We isolated lipopolysaccharide and capsular polysaccharide (K antigen)-defective mutants from two Klebsiella pneumoniaeparental strains, and compared their ability to colonize in vivothe germfree chicken gut. The high-molecular weight lipopolysaccharide (LPS) (O antigen) was found necessary for the colonization while the capsular polysaccharide (K2 or K29) was not of importance.

We obtained mutants devoid of the O1-antigen, the capsular polysaccharide (K antigen) or both fromKlebsiella pneumoniaeclinical isolates (urinary infection). These mutants were grown in urine, and their ability to fimbriate and to adhere were studied. Mutants lacking the O1-antigen, independently of the other surface molecules (capsule and fimbriae), showed a great decrease in adhesion to these cells.

ABSTRACTThe mechanism of killing of Aeromonas hydrophilaserum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic AeromonasO serogroups.

Bacteriophage PM4, PM5 and PM6 were isolated on different mesophilic Aeromonasstrains. These bacteriophage use the flagellum as their primary bacterial receptor since purified flagella from these strains are able to inactivate these bacteriophages, independently, and the phage‐resistant mutants are aflagellate and nonmotile. Furthermore, we showed that these bacteriophage may be useful to initiate the serotyping of mesophilic Aeromonasfor the H‐antigen (flagellum).

ABSTRACTWe have previously demonstrated the existence of Klebsiella pneumoniaeclinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates.

The following cell surface physicochemical characteristics were investigated inKlebsiella pneumoniae: surface charge, surface hydrophobicity by different methods, and accessibility of the lipid fraction of the outer membrane. The capsular polysaccharide, as well as the O-antigen repeating units of the lipopolysaccharide (LPS), conferred a hydrophilic, negatively charged surface to the bacterium, and a barrier to the dye congo red, which binds sites within the lipid fraction of the outer membrane (OM).

A study is presented to compare different methodologies for the cross-sectional visualization of membrane-electrode assemblies (MEAs) with SEM. Four techniques for cutting MEAs have been tested and analyzed: the sharp-edge cutting, CO2laser, embedded-mechanical polishing, and ion-milling. Characteristics of each procedure are compared. Sharp-edge cutting, being fast and low cost, may preserve layers porous morphology rather unaltered, but obtaining a good cross-section in a wide area is stochastic and with low probability. CO2-laser beam cutting alters severely carbon and Nafion layers as a consequence of the burning of carbon based materials. Embedded-mechanical polishing is a well stablished and reproducible method that shows accurately the thickness of the layers, however it is too aggressive with highly porous structures that may appear smoothed or removed. Ion-milling is also good for accurate thicknesses measurements, and more conservative with the porous morphologies than the previous one, but they are also smoothed.

We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cytitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.

Mesophilic Aeromonas hydrophila from serotypes O:11 and O:34 grown in a glucose-rich medium produce a capsule that can be seen under light and electron microscopy. The purified capsular polysaccharide has a composition qualitatively similar for strains O:11 and O:34, but quantitatively different. The capsular polysaccharides were immunogenic in rabbits, and did not cross-react with specific antibodies against either purified lipopolysaccharide from strains O:34 or O:11 or against the S-layer characteristic of strains from serotype O:11.

Bacteriophage PM4, PM5 and PM6 were isolated on different mesophilic Aeromonas strains. These bacteriophage use the flagellum as their primary bacterial receptor since purified flagella from these strains are able to inactivate these bacteriophages, independently, and the phage-resistant mutants are aflagellate and nonmotile. Furthermore, we showed that these bacteriophage may be useful to initiate the serotyping of mesophilic Aeromonas for the H-antigen (flagellum).

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.

Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37°C (serum-sensitive) but not when they were cultivated at 20°C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20°C and rough lipolysaccharide when cultivated at 37°C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.

PM3 is an Aeromonas-specific bacteriophage which was isolated and characterized on A. hydrophila strain TF7. Spontaneous mutants resistant to PM3 were non-motile having lost their characteristic monopolar flagellum. In addition, purified flagella inactivated PM3. PM3 is the first filamentous bacteriophage isolated on Aeromonas, the adsorption site for which is the monopolar flagellum.