Is reduced vancomycin susceptibility a factor associated with poor prognosis in MSSA bacteraemia?

OBJECTIVES: The known data about the influence of vancomycin MIC on Staphylococcus aureus bacteraemia are contradictory. Our objective was to study the possible impact of vancomycin MIC ≥1.5 mg/L on short- and medium-term mortality. METHODS: A prospective cohort study was carried out from March 2008 to January 2011 on adult patients with MSSA bacteraemia admitted to a tertiary hospital located in Seville (Spain). We studied the relationship between vancomycin MIC, accessory gene regulator (agr) type and absence of δ-haemolysin and poor prognosis. All isolates were genotyped by PFGE. Multivariate analysis, including a propensity score for having a vancomycin MIC of ≥1.5 mg/L, was performed by Cox regression. RESULTS: One hundred and thirty-five episodes of bacteraemia due to MSSA were included in the analysis. Twenty-nine (21.5%) isolates had a vancomycin MIC of ≥1.5 mg/L by Etest. There were no differences in agr distribution or absence of δ-haemolysin between isolates with reduced vancomycin susceptibility (RVS) and those without. RVS was not more frequent in specific clones; RVS was not associated with higher 14 or 30 day crude mortality (relative risk = 0.44, 95% CI = 0.14-1.35; and relative risk = 1.01, 95% CI = 0.52-1.96) rates, and it did not show higher rates of complicated bacteraemia (14.2% versus 13.8%, P = 0.61). Cox regression analysis did not significantly modify the results for 14 day mortality (HR = 0.39, 95% CI = 0.11-1.34) or 30 day mortality (HR = 0.89, 95% CI = 0.39-2.04). CONCLUSIONS: Contrary to previously published data, we did not find a relationship between RVS and higher mortality in patients with MSSA bacteraemia and we did not find a link with higher complicated bacteraemia rates.

In vitro effect photodynamic therapy with differents photosensitizers on cariogenic microorganisms.

BACKGROUND: Antimicrobial photodynamic therapy has been proposed as an alternative to suppress subgingival species. This results from the balance among Streptococcus sanguis, Streptococcus mutans and Candida albicans in the dental biofilm. Not all the photosensitizers have the same photodynamic effect against the different microorganims. The objective of this study is to compare in vitro the photodynamic effect of methylene blue (MB), rose Bengal (RB) and curcumin (CUR) in combination with white light on the cariogenic microorganism S. mutans, S. sanguis and C. albicans. RESULTS: Photodynamic therapy with MB, RB and CUR inhibited 6 log 10 the growth of both bacteria but at different concentrations: 0.31-0.62 µg/ml and 0.62-1.25 µg/ml RB were needed to photoinactivate S. mutans and S. sanguis, respectively; 1.25-2.5 µg/ml MB for both species; whereas higher CUR concentrations (80-160 µg/ml and 160-320 µg/ml) were required to obtain the same reduction in S. mutans and S. sanguis viability respectively. The minimal fungicidal concentration of MB for 5 log10 CFU reduction (4.5 McFarland) was 80-160 µg/ml, whereas for RB it ranged between 320 and 640 µg/ml. For CUR, even the maximum studied concentration (1280 µg/ml) did not reach that inhibition. Incubation time had no effect in all experiments. CONCLUSIONS: Photodynamic therapy with RB, MB and CUR and white light is effective in killing S. mutans and S. sanguis strains, although MB and RB are more efficient than CUR. C. albicans required higher concentrations of all photosensitizers to obtain a fungicidal effect, being MB the most efficient and CUR ineffective.

The coat protein is required for the elicitation of the Capsicum L2 gene-mediated resistance against the tobamoviruses.

In Capsicum, the resistance against tobamoviruses conferred by the L2 gene is effective against all but one of the known tobamoviruses. Pepper mild mottle virus (PMMoV) is the only virus which escapes its action. To identify the viral factors affecting induction of the hypersensitive reaction (HR) mediated by the Capsicum spp. L2 resistance gene, we have constructed chimeric viral genomes between paprika mild mottle virus (PaMMV) (a virus able to induce the HR) and PMMoV. A hybrid virus with the PaMMV coat protein gene substituted in the PMMoV-S sequences was able to elicit the HR in Capsicum frutescens (L2L2) plants. These data indicate that the sequences that affect induction of the HR mediated by the L2 resistance gene reside in the coat protein gene. Furthermore, a mutant that codes for a truncated coat protein was able to systemically spread in these plants. Thus, the elicitation of the host response requires the coat protein and not the RNA.

Rapid detection and differentiation of tobamoviruses infecting L-resistant genotypes of pepper by RT-PCR and restriction analysis.

A procedure involving reverse transcription followed by polymerase chain reaction (RT-PCR) was developed for typing pathotypes of the tobamoviruses infecting the L-resistant genotypes of pepper. The method provides a much simpler alternative to the bioassay tests for the different Capsicum spp. genotypes previously used. Discrimination between the two pathotypes, P1,2 and P1,2,3, which cannot be differentiated by serological means, was achieved by restriction enzyme analysis of the PCR products. The assay also detects and distinguishes both pathotypes in a single mixed-infected plant. The procedure should be useful for the diagnosis and control of the disease and helpful to breeders and biotechnologists when producing and evaluating resistance in pepper plants.

Accumulation, release and subcellular localization of azithromycin in phagocytic and non-phagocytic cells in culture.

The authors have examined the pharmacokinetic parameters of azithromycin in phagocytic (J774 macrophages) and non-phagocytic (rat embryo fibroblasts and NRK-cells) cultured cells. Azithromycin demonstrates an exceptionally large accumulation in all the cell types tested (perhaps in two functionally and structurally distinct compartments) and a slow release of the cell-associated drug. Azithromycin probably accumulates in cells by a non-specific transport process following the model of diffusion/segregation. The cell-associated drug distributes mostly in the lysosomal compartment (50-70%) and the remaining part is freely soluble in the cytosol. In fibroblasts, and to a lesser extent in NRK-cells, azithromycin (10mg/l) induces a decrease of the buoyant density of the lysosomes which may be brought about by the drug itself together with osmotically-bound water and/or by the accumulation of low-density materials within these organelles. These observations open important questions with respect to the potential toxicity of azithromycin. The significance of such alterations and of their biological consequences are at present under investigation.

Altered local and systemic spread of movement deficient virus in transgenic tobacco plants expressing the cucumber mosaic virus 3a protein.

The 3a protein encoded by RNA 3 of cucumber mosaic virus has been identified as the viral cell-to-cell movement protein. The constitutive expression in transgenic tobacco plants of 3a protein from a subgroup I strain was able to complement in trans the short distance movement of a 3a defective CMV mutant belonging to a different taxonomic subgroup. This ability was dependent upon the accumulation levels of the 3a protein in transgenic tobacco plants. However, an initial delay in viral accumulation and spread of the defective virus as compared to the wild type virus was determined in complementation tests. Furthermore, a reduction in disease symptoms as well as a different pattern of systemic viral distribution from those of the wild type virus was detected. These results show that the early events in viral infection affect the long distance spread of the virus. Finally, the wild type virus moved faster in the 3a protein-expressing plants than in control plants, thus indicating that the constitutive expression of the 3a protein favours long-distance viral spread.

The coat protein of tobamovirus acts as elicitor of both L2 and L4 gene-mediated resistance in Capsicum.

In Capsicum, the resistance conferred by the L(2) gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L(4) gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L(2) gene confers resistance to Paprika mild mottle virus (PaMMV), while the L(4) gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L(2)L(2)) and Capsicum chacoense (L(4)L(4)) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L(2)L(2)) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L(2) resistance gene. In C. chacoense (L(4)L(4)) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L(4) gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L(2)L(2)) leaves and PMMoV CP into C. chacoense (L(4)L(4)) leaves by biolistic co-bombardment with a beta-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L(2) and L(4) gene-mediated hypersensitive response.

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression.

Resistance to pepper mild mottle tobamovirus conferred by the 54-kDa gene sequence in transgenic plants does not require expression of the wild-type 54-kDa protein.

We previously reported that Nicotiana benthamiana plants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV-S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenllado et al., 1995, Virology 211, 170--183). Here we show that some of the N. benthamiana plants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild-type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype.

Accumulation kinetics of CMV RNA 3-encoded proteins and subcellular localization of the 3a protein in infected and transgenic tobacco plants.

The complete nucleotide sequence of RNA 3 of a Spanish isolate of cucumber mosaic virus (CMV-24) has been determined. The encoded putative cell-to-cell movement protein (3a protein) and the coat protein are 279 and 218 amino acids long, respectively. The 3a protein was expressed in Escherichia coli using the vector pT7-7 and was used to raise an immunoserum. We have followed the time course of accumulation of the 3a protein, in parallel to that of the coat protein, and its subcellular localization as a function of time after CMV-24 infection on tobacco plants. The maximum accumulation level of the 3a protein was reached at early stages of infection, being detected in the cytosolic and the cell wall fractions. At later stages of infection, a decline in accumulation levels of the 3a protein was observed, and the protein was essentially associated with the cell wall fractions. These data were corroborated by immunocytochemistry performed in both infected and 3a-expressing transgenic tobacco plants.

Isolation and partial characterization of a monoclonal antibody specific for a naturally occurring double-stranded RNA.

A monoclonal antibody (Mab) against the double-stranded form of the cucumber mosaic virus-associated RNA 5, the sequence variant that induces the tomato necrosis disease (ds(n)CARNA 5), has been obtained. Specificity studies using a radioimmunoassay showed that the purified Mab recognizes neither DNA nor ssRNA of different sources including ss(n)CARNA 5, poly(I).poly(C), nor dsRNA of two other sequence variants of CARNA 5. Therefore, the Mab seems to recognize a conformational variation only present in the double helical structure of ds(n)CARNA 5.

Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection.

Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus (PMMV) replicase gene were generated and six independently transformed plant lines were analyzed for resistance to PMMV. Two different resistance responses were obtained. Some of the transgenic plants from only two lines showed a preestablished, complete, and highly resistant phenotype since no viral symptoms were observed, although a low level of virus replication occurred. The remaining plants from these two lines and all of the plants from the other four lines tested showed a delayed, induced, and also highly resistant phenotype since they were susceptible early, but were able to recover from the systemic PMMV infection. Recovered, symptomless leaves were resistant to the PMMV strain from which the 54-kDa gene was derived and to a closely related strain but not to tobacco mosaic virus. Such a delayed resistance phenotype has not been previously described for any plant expressing viral replicase sequences. The transgenic plants within the lines displaying complete or delayed resistance phenotypes were analyzed for transgene expression before and after PMMV inoculation and the two types of resistance responses were shown to be independent of the transgene transcript level.

Nucleotide sequences of 5' and 3' non-coding regions of pepper mild mottle virus strain S RNA.

The nucleotide sequences of the 5' and 3' non-coding regions of pepper mild mottle virus strain S (PMMV-S) RNA were determined; they are more like corresponding sequences of tomato mosaic virus (ToMV) RNA than those of any other tobamovirus reported so far. The 5' leader contains a 68 nucleotide guanosine-free sequence which differs in several nucleotides from the corresponding sequences in genomic RNA of tobacco mosaic virus (TMV) and ToMV. The messenger activity of PMMV-S RNA in vitro and the polypeptide translation products made were similar to those of TMV RNA. It therefore seems unlikely that qualitative or quantitative differences in translation in vivo account for the milder symptoms induced by PMMV-S, and its lesser replication, than TMV. The 3' non-coding region of PMMV-S RNA is 199 nucleotides long and can be folded into the same secondary structure as the RNA of other tobamoviruses.

The 3a protein from cucumber mosaic virus increases the gating capacity of plasmodesmata in transgenic tobacco plants.

The 3a protein, encoded by RNA 3 of cucumber mosaic virus (CMV), is the putative movement protein of viral progeny in infected plants. An analysis of transgenic tobacco plants constitutively expressing the CMV 3a protein showed that the protein is accumulated in leaves at every stage of development. In fully expanded leaves the protein is immunodetectable mostly in a cell-wall-enriched fraction. Dye-coupling experiments using fluorescent-dextran probes were performed on fully expanded leaves to study the modifying effect of CMV 3a protein on the gating capacity of plasmodesmata. Movement of fluorescein-isothiocyanate-labelled dextran with a mean molecular mass of 10,000 Da, and an approximate Stokes' radius of 2.3 nm, was detected between cells of the 3a protein transgenic plants, but not in the control plants. These results are consistent with the idea that the CMV 3a protein is involved in the modification of plasmodesmata and, therefore, in the cell-to-cell spread of the virus.

Biological and molecular characterization of P101 isolate, a tobamoviral pepper strain from Bulgaria.

A tobamovirus isolated from pepper crops in Bulgaria has been characterized, and is referred to below as P101. It was closely related to Paprika mild mottle virus (PaMMV) (Dutch isolate), based upon the serological relationship of its coat protein, and the nucleotide sequence analysis of the gene encoding the coat protein and the 3' non-coding region of the viral RNA. The coat proteins of the two isolates differ by two amino acids, and these substitutions may be responsible for the different reactivity of the isolates towards a polyclonal antiserum raised against the virion of the Dutch isolate. The biological behaviour of both isolates was similar in the hosts tested, except in pepper plants where P101 induced delayed and milder symptoms compared with PaMMV, although their accumulation levels were similar. In addition, we investigated the infection pattern of the two isolates in tomato plants. Both isolates accumulated in protoplasts as well as in inoculated leaves, although systemic invasion was limited. This limited spread was not due to activation of defense mechanism(s) in the plant, since the upper uninoculated leaves from P101-infected tomato plants were fully susceptible to challenge inoculation with the virus. Instead, it appears due to a restriction of long-distance movement, that could be overcome in tomato plants co-infected with Tobacco mosaic virus (TMV), but not with either Cucumber mosaic virus or Pepino mosaic virus. The ability of P101 to move systemically in the presence of TMV was not linked to enhanced accumulation of P101 at the cellular level. Thus, a tobamovirus but not the viruses tested from other genera could complement, in trans, the function(s) required for PaMMV to invade the upper uninoculated leaves. Paprika mild mottle virus strain B is proposed as the name for this new isolate.

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper.

The entire genomic RNA of a Spanish isolate of pepper mild mottle virus (PMMV-S), a resistance-breaking virus in pepper, was cloned and sequenced and shown to be similar to other tobamoviruses in its genomic organization. It consisted of 6357 nucleotides (nt) and contained four open reading frames (ORFs) which encode a 126K protein and a readthrough 183K protein (nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a 17.5K coat protein (nt 5685 to 6158). This is the first tobamovirus in which none of the ORFs overlap. Both its nucleic acid and predicted protein sequences were compared with the previously determined sequences of other tobamoviruses. The variations and similarities found and their relationship with the pathogenicity of this virus are discussed.

The Capsicum L3 gene-mediated resistance against the tobamoviruses is elicited by the coat protein.

The L3 gene is responsible for the hypersensitive response in Capsicum plants against infection by tobamoviruses. The resistance conferred by this gene is one of the most effective so far described against tobamoviruses. Certain isolates of pepper mild mottle virus (PMMV) are the only tobamoviruses able to overcome the L3 resistance. Chimeric viral genomes between PMMV-S (to which L3 plants are hypersensitive) and PMMV-I (an L3 resistance-breaking isolate) led us to conclude that sequence variation within the coat protein gene of both isolates determines their different virulence in L3L3 plants. Furthermore, the results indicate that a single amino acid substitution, Asn to Met, at position 138 of the PMMV-I coat protein is sufficient to induce the hypersensitive response and localization of viral infection in C. chinense plants. Finally, the use of a mutant coding for a truncated coat protein (maintaining the Met138 coding sequence at the RNA level) demonstrates that a functional coat protein is required for elicitation of the L3 gene-mediated resistance.

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses.

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3' non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3' non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed.

Interaction of aminoglycosides and cephalosporins against Pseudomonas aeruginosa. Correlation between interaction index and killing curve.

Three multiresistant clinical isolates of Pseudomonas aeruginosa were evaluated, by the chequerboard and the killing curve methods, for in-vitro synergy between cephalosporins and aminoglycosides. The killing-curve method was standardized to give results comparable with those obtained with the chequerboard test (FIC index). This was achieved with combinations showing synergy by chequerboard (FIC less than or equal to 0.75) by using in the killing curves subinhibitory concentrations: half the MIC in single antibiotic assay and one eighth the MIC in the combinations. For combinations that showed indifference by chequerboard (FIC = 1) half the MIC was used for antibiotics alone and in combination. For the antagonistic combinations by chequerboard (FIC greater than 2) concentrations equal to the MIC were used with single antibiotics and combinations in the killing curve experiments. Prediction of killing curve results could then be obtained with the FIC index. The killing curve results could not be explained by pH changes or inactivation of the antibiotics.