Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Broilers Throughout the Supply Chain in Valencia, Spain.

Campylobacter is a major foodborne pathogen and its antimicrobial resistance (AMR) has been described worldwide. The main objective of this study was to determine the occurrence and AMR of Campylobacter spp. isolated from broilers throughout the supply chain in Valencia, Spain. A total of 483 samples were included in the analysis: 430 from the slaughterhouse (chicken carcass and neck skin) and 53 from the point of sale (retail broiler and packaging). Taking into account the origin of the sample, the prevalence of Campylobacter spp. was 19% in carcass, 28.2% in neck skin, 36.7% in retail broiler, and 80% in packaging isolates. The prevalence of different species in the analyzed samples was 21.1% and 4.8% for Campylobacter jejuni and Campylobacter coli, respectively. AMR profiling of 125 Campylobacter isolates revealed that 122 (97.6%) of the isolates were resistant to one or more antimicrobials. C. jejuni samples presented high resistance to nalidixic acid and ciprofloxacin, 96.1% and 90.2% respectively, whereas C. coli showed 87% of resistance to both antimicrobials. Both species were resistant to tetracycline (C. jejuni 84.3% and C. coli 60.9%) and 26.1% of C. coli was resistant to streptomycin. These results showed no significant difference in the frequency of AMR (p ≥ 0.05) among isolates originated from different points in the food-processing chain at slaughterhouses and retail establishments. In contrast, three main patterns were detected: quinolone-tetracycline (64%), quinolone-only (17.6%), and quinolone-tetracycline-aminoglycosides (8%). Additionally, 12.8% of the isolates presented multidrug resistance, with significantly higher levels detected among C. coli (30.4%) isolates compared with C. jejuni (8.8%) and all the three strains were resistant to all six antibiotics tested. Therefore, these results indicate that broilers could be a source of antimicrobial-resistant Campylobacter in humans and consequently pose a risk to public health.

Analysis of pharmaceutical biodegradation of WWTP sludge using composting and identification of certain microorganisms involved in the process.

Pharmaceuticals (PhCs) are organic contaminants that have been detected in wastewater, surface water, and soils throughout the world. The presence of 10 commonly used PhCs in Spain (azithromycin, benzylpenicillin, citalopram, fluconazole, fluoxetine, ibuprofen, irbesartan, olanzapine, telmisartan, and venlafaxine) was analysed at four wastewater treatment plants, and the changes in their concentrations during treatment were assessed. Although certain some PhCs were degraded in the treated water, their presence in sewage sludge increased in all cases. The sewage sludge was composted using rice straw to degrade the PhCs, and the composting efficiency was modified by changes in the relative C/N ratio of the composting blend. Using a simple microbiological culture process for enrichment, 11 different strains of microorganisms that degraded specific PhCs were identified. Ibuprofen and azithromycin were metabolized by one and four strains, respectively, and both PhCs were used as a carbon source; in addition, six strains used irbesartan as a nitrogen source.

The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection.

Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here the phenotypic effects produced by the inhibition of transglutaminase enzymatic activity with cystamine, including growth inhibition of yeast cells, induction of autophagy in response to damage caused by cystamine, alteration of the normal yeast division pattern, changes in cell wall, and inhibition of the yeast-to-mycelium transition. The latter phenomenon was also observed in the C. albicans ATCC 26555 strain. Growth inhibition by cystamine was also determined in other Candida strains, demonstrating the importance of transglutaminase in these species. Finally, we identified enolase 1 as the cell wall protein responsible for TGase activity. After studying the inhibition of enzymatic activities with anti-CaEno1 antibodies and through bioinformatics studies, we suggest that the enolase and transglutaminase catalytic sites are localized in different domains of the protein. The aforementioned data indicate that TGase/Eno1 is a putative target for designing new drugs to control C. albicans infection.

Bassianolone: an antimicrobial precursor of cephalosporolides E and F from the entomoparasitic fungus Beauveria bassiana.

We have established the chemical structure of (+)-bassianolone (3), the antimicrobial compound precursor of cephalosporolides E and F, and that of the furan metabolite 4 from the entomopathogenic fungus Beauveria bassiana.

Characteristics of rice straw and sewage sludge as composting materials in Valencia (Spain).

This work supports the idea that composting can be useful for minimizing the rice straw and sewage sludge environmental impact. Several physical, chemical and microbiological properties of these raw materials were analyzed. The characteristics of the rice straw were complementary to those of the sewage sludge for the application of composting. The C/N ratios suitable for a rapid increased in microbial activity were the lowest (17-24). A temperature of 62 degrees C during 48 h removed pathogenic microorganisms from rice straw and sewage sludge mixture. The results obtained in the present work suggested that these materials could be use in the composting process.

The use of trypsin to solubilize wall proteins from Candida albicans led to the identification of chitinase 2 as an enzyme covalently linked to the yeast wall structure.

The use of trypsin to break proteins covalently linked to the yeast walls of Candida albicans released approx. 50% of the proteins, but also glucose and N-acetylglucosamine. Analysis by affinity chromatography indicated that glucose and/or N-acetylglucosamine formed part of the same supramolecular complexes with mannoproteins. These complexes would represent a new type of cell wall structuration in which beta-1,6 glucan and chitin are linked to proteins. An internal peptide from a 50-kDa protein released by trypsin was sequenced, showing 100% identity with chitinase 2 protein and 92% with chitinase 3. The electrophoretic mobility of the chitinase 2 protein was changed by treatment with EndoH or beta-elimination, indicating that the enzyme was both N- and O-mannosylated.

Transglutaminase activity is involved in Saccharomyces cerevisiae wall construction.

Transglutaminase activity, which forms the interpeptidic cross-link N(epsilon)-(gamma-glutamyl)-lysine, was demonstrated in cell-free extracts of Saccharomyces cerevisiae by incorporation of [(14)C]lysine into an exogenous acceptor, N,N'-dimethylcasein. Higher levels of the activity were present in the cell wall, which also contained endogenous acceptors. The enzyme activity in the wall was inhibited by cystamine, a known inhibitor of transglutaminase, and by EDTA, indicating a cation-dependent activity. After the endogenous wall acceptors were labelled radioactively by transglutaminase, extraction with SDS solubilized about 50% of the total radioactivity, while Zymolyase and chitinase each released a further 3%. The proteins solubilized by SDS had molecular masses less than 50 kDa, whereas the material released by Zymolyase or chitinase had molecular masses greater than 180 kDa, suggesting a precursor-product relationship. Cystamine inhibited the growth of several strains of S. cerevisiae. Treated cells showed increased sensitivity to Zymolyase and appeared as protoplasts, indicating gross alterations in the cell wall. These data suggest that transglutaminase may be involved in the formation of covalent cross-links between wall proteins during wall construction.

The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis.

Saccharomyces cerevisiaepresents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiaeW27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiaeW27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2gene acts as a negative regulator of pseudohyphal growth.

Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.

The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls.

Monoclonal antibody 3H8: a useful tool in the diagnosis of candidiasis.

In a previous series of experiments six mAbs were obtained against cell wall extracts of Candida albicans ATCC 26555. After several studies only one of them, designated 3H8, has been used to produce a commercial kit for the rapid diagnosis of candidiasis, Bichro-latex albicans (Fomouze Diagnostics). The present study involved the generation and characterization of this mAb as an immunoglobulin G1 which recognizes mannoproteins of high molecular mass present in the C. albicans cell wall. ELISA assays showed that the presence of the epitope recognized by mAb 3H8 was similar in both yeast and mycelial cell walls of C. albicans, in contrast to the epitope for mAb 1B12, which is mainly expressed in the yeast cell wall. The 3H8 epitope was located at the external surface in C. albicans ATCC 26555, whereas it is partially cryptic in the cell wall in other C. albicans strains. No reaction was observed with other Candida species. Immunohistochemical studies using this antibody demonstrated that it specifically recognized C. albicans in tissue, detecting mycelial forms and, to a lesser extent, blastospores, suggesting that it is also a valuable tool in the evaluation of fungal infections in paraffin-embedded tissue, particularly when identification is required.