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Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xcc&phi, 1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium which colonizes the human stomach. The ability of H. pylori to evade the host defense system and the emergence of antibiotic resistant strains result in bacteria persistence and chronic inflammation, which leads to both severe gastric and extra-gastric diseases. Consequently, innovative approaches able to overcome H. pylori clinical outcomes are needed. In this work, we develop a novel non-toxic therapy based on the synergistic action of H. pylori phage and lactoferrin adsorbed on hydroxyapatite nanoparticles, which effectively impairs bacteria colonization and minimizes the damage of the host pro-inflammatory response.

A flow cytometry technique that unequivocally identifies some of the toxic metabolites produced by Phaeomoniella chlamydospora, one of the main fungal pathogens causing esca disease of grapevine (Vitis vinifera), was developed. Antibodies raised against exopolysaccharides (EPS)—metabolites produced by Pa. chlamydospora that have been reported to be phytotoxic—were used as antigen to immunize rats. The specificity of these antibodies was assayed by flow cytometry against Pa. chlamydospora polysaccharides and against EPS with a different structure isolated from other phytopathogenic fungi, including Phaeoacremonium aleophilum and the Botryosphaeriaceae species Neofusicoccum luteum and N. parvum. Using this method, Pa. chlamydospora polysaccharides were detected in the symptomatic leaves of esca-affected grapevines, while healthy and asymptomatic leaves from both healthy and diseased vines did not produce a binding reaction. This method potentially could be used to develop a simple kit to study the mechanisms underlying the development of esca foliar symptoms and to indirectly assess the presence of Pa. chlamydospora in grapevine material.

Six hundred fourteen strains of Salmonella enterica were isolated from 16,926 samples of food of animal origin collected in southern Italy from 2003 to 2012. The isolates were identified, serotyped, and challenged against 15 antibiotics according to the protocol defined at national level for veterinary isolates of Salmonella (EnterVet surveillance network). Salmonella serotypes Typhimurium, Hadar, Enteritidis, Derby, and 4,[5],12:i:- were those most frequently isolated. The widest resistances were recorded towards sulfonamides (69 % of the isolates), trimethoprim-sulfamethoxazole (52 % of the isolates), and tetracycline (51 % of the isolates). The rate of multidrug resistance of the isolates decreased significantly from the first 5 years of the study period (82.6 %) to the last 5 years (54.3 %).

Non-typhoidal Salmonella enterica infection is a significant public health problem worldwide. The aim of this study was to characterize Salmonella enterica strains isolated from human specimens in central and southern Italy, for epidemiological studies. One hundred and fifty S. enterica strains were serotyped. Isolates were tested for their antimicrobial susceptibility, by disk diffusion method. The molecular characterizations, based on PCR, were carried out for the detection of invA gene and other virulence elements and phage marker genes. Eighteen different Salmonella serotypes were identified. The most common serotypes detected were S. Typhimurium, S. Enteritidis, the monophasic variant of S. Typhimurium (S. 4,[5],12:i:-), and S. Napoli. High resistance rates were recorded for tetracycline (64%), streptomycin (62%), sulphonamide (57%), and ampicillin (56%). The ASSuT R-type, also associated to resistance to other antibiotics, was highly prevalent in S. 4,[5],12:i:- (97%) and S. Typhimurium (55%), while the ACSSuT Rtype, also associated to other antibiotics, was observed prevalently in S. Typhimurium (20.4%). The genes of more common detection were invA (100%), sspH2 (86.6%), gtgB (84.6%), g8 (80%), sodC1 (77.3%), gipA (52.6%), sspH1 (52.6%).

In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

The modelling of peptidoglycan is responsible for key cellular processes in Mycobacterium tuberculosis such as cell growth, division and resuscitation from dormancy. The structure of M. tuberculosis peptidoglycan is atypical since it contains a majority of 3,3 cross-links synthesized by L,D-transpeptidases that replace the 4,3 cross-links formed by the D,D-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these L,D-transpeptidases and in combination with clavulanic acid are bactericidal against extensively drug-resistant M. tuberculosis. Here, crystal structures of the L,D-transpeptidase Ldt(Mt1) from M. tuberculosis in a ligand-free form and in complex with the carbapenem imipenem are reported. Elucidation of the structural features of Ldt(Mt1) unveils analogies and differences between the two key transpeptidases of M. tuberculosis: Ldt(Mt1) and Ldt(Mt2). In addition, the structure of imipenemin-activated Ldt(Mt1) provides a detailed structural view of the interactions between a carbapenem drug and Ldt(Mt1). By providing the key interactions in the binding of carbapenem to Ldt(Mt1), this work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antitubercular agents against drug-resistant M. tuberculosis.

Background Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. Methods Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB-G6A sequence, which was further optimized by increasing the peptide positive charge (TB-KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. Results TB-KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. Conclusion Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB-KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. General significance The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB-KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process. © 2013 Elsevier B.V. Background Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. Methods Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB-G6A sequence, which was further optimized by increasing the peptide positive charge (TB-KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. Results TB-KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. Conclusion Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB-KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. General significance The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB-KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process. © 2013 Elsevier B.V.

There is epidemiological evidence that H. pylori might predispose to Alzheimer’s disease. To understand the cellular processes potentially linking such unrelated events, we incubated the human gastric cells MNK-28 with the H. pylori peptide Hp(2-20). We then monitored the activated genes by global gene expression. The peptide modulated 77 genes, of which 65 are listed in the AlzBase database and include the hallmarks of Alzheimer’s disease: APP, APOE, PSEN1, and PSEN2. A large fraction of modulated genes (30 out of 77) belong to the inflammation pathway. Remarkably, the pathways dis-regulated in Alzheimer’s and Leasch-Nyhan diseases result dis-regulated also in this study. The unsuspected links between such different diseases – though still awaiting formal validation – suggest new directions for the study of neurological diseases.

The phytotoxic exopolysaccharides produced by Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, were isolated and partially identified. Their phytotoxic activity was evaluated on host and non-host plants and their role in the complex mechanisms of host-pathogen interaction was also discussed. The phytotoxic exopolysaccharides, which are natural antigens, were used to arise specific antibodies by rat immunization. The antibodies were used to develop a rapid and specific method to unambiguously detect P.s. pv. actinidiae exopolysaccharides isolated from bacterial culture and infected plant samples. Indeed, the antibodies recognized the exopolysaccharides produced by other two strains of P. s. pv. actinidiae but did not cross reacted with those isolated from P. s. pv. syringae and Pseudomonas viridiflava culture filtrates. Finally, the same antibodies significantly recognized the exopolysaccharides extracted from infected kiwi leaves.

Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortusantibodies (by the agglutination and complement fixation tests) and theNramp1genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and theNramp1genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in theNramp1A+(Nramp1AAorNramp1AB) group and 6 out of 64 (9.4%) in theNramp1A−(Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; χ2, 11.65 for 1 degree of freedom). Monocytes fromNramp1BBsubjects displayed significantly (P< 0.01) higher levels ofNramp1mRNA thanNramp1AAsubjects and also a significantly (P< 0.01) higher ability in controlling the intracellular replication of severalBrucellaspecies in vitro. Thus, selection for theNramp1BBgenotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.

Nunzia Nocerino,1 Andrea Fulgione,1 Marco Iannaccone,1 Laura Tomasetta,1 Flora Ianniello,1 Francesca Martora,1 Marco Lelli,2 Norberto Roveri,2 Federico Capuano,3 Rosanna Capparelli1 1Department of Agriculture Special Biotechnology Center Federico II, CeBIOTEC Biotechnology, University of Naples Federico II, Naples, 2Department of Chemistry, G Ciamician, Alma Mater Studiorum, University of Bologna, Bologna, 3Department of Food Inspection IZS ME, Naples, Italy Abstract: The emergence of bacterial strains resistant to antibiotics is a general public health problem. Progress in developing new molecules with antimicrobial properties has been made. In this study, we evaluated the biological activity of a hybrid nanocomposite composed of synthetic biomimetic hydroxyapatite surface-functionalized by lactoferrin (LF-HA). We evaluated the antimicrobial, anti-inflammatory, and antioxidant properties of LF-HA and found that the composite was active against both Gram-positive and Gram-negative bacteria, and that it modulated proinflammatory and anti-inflammatory responses and enhanced antioxidant properties as compared with LF alone. These results indicate the possibility of using LF-HA as an antimicrobial system and biomimetic hydroxyapatite as a candidate for innovative biomedical applications. Keywords: lactoferrin, hydroxyapatite nanocrystals, biomimetism, biological activity, drug delivery

The virulence of bacteria can be evaluated through both phenotypic and molecular assays. We applied these techniques to 114 strains of Salmonella enterica subsp. enterica collected from July 2010 to June 2012. Salmonella strains were of human origin (71/114) or isolated from food (43/114). The strain set included only the three predominant Salmonella serovars isolated in Italy from humans (S. Enteritidis, S. Typhimurium, S. 4,[5],12:i:-). These strains were screened via polymerase chain reaction for 12 virulence factors (gipA, gtgB, sopE, sspH1, sspH2, sodC1, gtgE, spvC, pefA, mig5, rck, srgA), while antimicrobial sensitivity was evaluated through the Kirby-Bauer assay. Fifty-nine different virulence profiles were highlighted; the genes showing the highest homology were those related to the presence of prophages (gipA, gtgB, sopE, sspH1, sspH2, sodC1, gtgE), while the genes related to the presence of plasmids were less frequently detected (spvC, pefA, mig5, rck, srgA). The Salmonella serovars Typhimurium and 4,[5],12:i:- were closely related in terms of both virulotyping and antibiotic resistance. S. Enteritidis showed higher antibiotic sensitivity and a higher prevalence of genes related to plasmids.

Toll-like receptors recognize several components of Mycobacterium tuberculosis, the main causative agent of tuberculosis. The signaling pathways leading to activation of the immune response require the MyD88 and TIRAP genes. The hypothesis that polymorphic variants of these genes influenced resistance to pulmonary tuberculosis was tested by a case-control study (400 cases and 400 controls). Heterozygosity at the polymorphic sites MyD88 rs6853 (alleles: A, G) or TIRAP rs8177374 (S180L) (alleles: C, T) is associated With resistance to pulmonary tuberculosis (P: 7.8 x 10(-8) and 2 x 10(-6), respectively). Double heterozygosity confers higher protection levels (P: 10(-14) to 2 x 10(-16)). The logistic regression model displayed that the double homozygous genotype GG/TT predisposes to the disease (odds ratio (OR): 5.78) and the AG/TT genotype combination neutralizes the protective activity. exerted by AG (OR: 3.05). The same model showed that the risk of developing the disease increases with age from 31-40 years to 71-80 years (OR: 1.32-13.59).

Background The resistance of Helicobacter pylori to the antibiotic therapy poses the problem to discover new therapeutic approaches. Recently it has been stated that antibacterial, immunomodulatory, and antioxidant properties of lactoferrin are increased when this protein is surface-linked to biomimetic hydroxyapatite nanocrystals. Objective Based on these knowledge, the aim of the study was to investigate the efficacy of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles with cell free supernatant from probiotic Lactobacillus paracasei as an alternative therapy against Helicobacter pylori infection. Methods Antibacterial and antinflammatory properties, humoral antibody induction, histopathological analysis and absence of side effects were evaluated in both in vitro and in vivo studies. Results The tests carried out have been demonstrated better performance of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles combined with cell free supernatant from probiotic Lactobacillus paracasei compared to both lactoferrin and probiotic alone or pooled. Conclusion These findings indicate the effectiveness and safety of our proposed therapy as alternative treatment for Helicobacter pylori infection.

The use of molecular markers for fingerprinting applied to genotype represents a useful tool for the traceability of production in the agri-food chain and for the identification and control of propagation material during the various nursery stages. Apricot takes an important place in Campania fruit production. The aim of this work is to make a contribution to complete the characterization of local (Campania Region) apricot germplasm and to show that the analysis of DNA by molecular markers is a useful tool for the defence of typical local products. In this work, 10 apricot cultivars were analyzed by performing Random Amplified Polymorphic DNA (RAPD)-PCR. RAPDs are based on PCR amplification of polymorphic DNA using single primers containing arbitrary nucleotide sequences. Our results show that 8 cultivars present a unique profile of amplification, thus providing a fingerprint of their genome.

Background The most serious criticisms leveled at bacteriophage therapy are as follows: phages induce neutralizing antibodies, phages are active only when administered shortly after bacterial infection, and phage-resistant bacteria emerge rapidly in the course of therapy. Methods Phages lytic for several Salmonella enterica serovars were isolated by means of standard protocols from feces of patients with gastroenteritis. Growth of S. enterica serovar Paratyphi B (Salp572(phi1S)) in the presence of phage phi1 (selected from among 8 phages for its larger host range) provided a phage phi1-resistant bacterial strain (Salp572(phi1R)). The properties of the Salp572(phi1S) and Salp572(phi1R) strains and of phage phi1 were studied in a mouse model of experimental infection. Results Phages induced nonneutralizing antibodies and were active 2 weeks after experimental infection of mice; phage-resistant bacteria were avirulent and short lived in vivo. More importantly, phage-resistant bacteria were excellent vaccines, protecting against lethal doses of heterologous S. enterica serovars. Conclusions Phage therapy effectiveness has not yet been properly assessed.

Puroindoline A and puroindoline B from plant seeds, bovine lactoferrin and chicken eggs lysozyme are antimicrobial proteins of innate immune system that lyse invading organisms. We investigate their potential antibacterial activity against Listeria monocytogenes in a mouse model. Bacteria were isolated from various organs for 7 days after challenge. Livers displayed consistently higher bacterial count (up to 107 cfu/g) than spleens, kidneys and brains. The efficacy of the AMPs was therefore established by measuring the infection level (cfu number) of these organs. Puroindoline A and puroindoline B (5 mg/mouse), lactoferrin and lysozyme (1.25 mg/mouse), intravenously injected individually, inhibited bacterial growth completely. Puroindoline A, puroindoline B and lactoferrin were effective when administered 24 h before infection; lysozyme was effective at the time of infection or 5 days after. Their combined use resulted in the enhancement of individual antibacterial activities. Complete inhibition of bacterial growth was observed using concurrently 0.059 mg/mouse of puroindoline A and 0.019 mg/mouse of puroindoline B, lactoferrin and lysozyme. Individual antimicrobial proteins reduced significantly the expression level of pro-inflammatory cytokines (IL-6, IL-8, INF-γ and TNF-α), acute phase proteins (C-reactive protein and fibrinogen) and the T lymphocyte antigens CD4, CD8a, CD8b and CD25. These results suggest their potential use for the control of L. monocytogenes infections.

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

AIMS: To ascertain whether in Brucella abortus-infected water buffalo herds, the number of newly infected animals could be reduced by culling superspreaders (the animals secreting > or =10(4) CFU per ml of milk). METHODS AND RESULTS: The number of B. abortus present in the milk (CFU per ml) from 500 water buffaloes was measured by the culture. Each animal was tested three times, at one month intervals. The presence or the absence of B. abortus in each milk sample was confirmed by PCR. A majority of infected animals shed the pathogen at a low level (< or =10(3) CFU ml(-1)). However, a few infected individuals (superspreaders) shed large numbers of B. abortus (> or =10(4) CFU ml(-1)). Quantitative PCR of B. abortus positive milk samples gave comparable results to culture. Culling of the superspreaders was sufficient to arrest the spread of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach described here can reduce significantly the cost of controlling brucellosis. Culture and quantitative PCR tests identify superspreaders and, compared with the serological tests in use to detect brucellosis, provide also a more accurate estimate of the disease incidence.

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10–14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3–6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

Lactoferrin (LF) is a member of the transferrin family of iron-binding glycoproteins. It is also a multifunctional protein of 80 kDa that is synthesized by glandular epithelial cells and secreted into mucosal fluid. High levels of LF are present in colostrom and milk and low levels in tears, saliva, and gastrointestinal and reproductive secretions. Data regarding the antifungal effects of LF are limited. Studies have been performed on Candida albicans, which demonstrated that LF inhibits the growth of this fungus. This study reports the results of experiments carried out in order to evaluate the effects of LF on the growth of 11 fungi, which were isolated from plants and soils. These experiments employed the methods of amended agar utilizing nine different concentration levels of LF (0, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 5000 mg L(-1)). The effects of LF on the growth of these fungi were based on measures of the radial growth of the fungal colonies expressed both as percentage of inhibition and as IC(50) values (the concentration at which the fungal growth was inhibited by 50\% relative to controls). LF had no effects on Alternaria alternata, Gliocladium roseum, Fusarium solani and Colletotrichum lindemuthianum. It did, however, inhibit the growth of Aspergillus niger, Trichoderma viride, Sclerotinia sclerotiorum, Sclerotium rolfsii, Rhizoctonia solani and Phoma exigua to the point that their IC(50) values ranged from 31.1 mg L(-1) for S. sclerotiorum to 952 mg L(-1) for T. viride.

The present study describes a bacteriophage (M Sa ) active against Staphylococcus aureus , including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (10 8 CFU/mouse), phage (10 9 PFU) rescued 97% of the mice; when applied to nonlethal (5 × 10 6 CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M Sa , delivered inside macrophages by S. aureus , kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human S. aureus infections.

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortusantibodies (by the skin test, the agglutination test, and the complement fixation test) and theNramp1genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3′ untranslated region of theNramp1gene. TheBBgenotype was represented among only controls, providing evidence that this genotype confers resistance toBrucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level ofNramp1mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.

The 3′ untranslated region of the water buffalo Nramp1 (natural resistance-associated macrophage protein 1) gene contains two alleles (Nramp1A and Nramp1B), as detected by the denaturing gradient gel electrophoresis (DGGE) technique. The Nramp1BB genotype is associated with resistance of water buffalo to the intracellular pathogen Brucella abortus. This article provides evidence that the Nramp1AA genotype is associated with susceptibility to the same pathogen. Susceptibility or resistance of water buffalo to B. abortus was established by agglutination, complement fixation, and skin tests. The Nramp1 genotype was established by DGGE analysis. The association between the Nramp1AA genotype and susceptibility to B. abortus was demonstrated in two independent population samples (152 cases and 281 controls; 87 cases and 124 controls, respectively). Macrophages from Nramp1AA subjects displayed a lower Nramp1 mRNA level when compared with macrophages from Nramp1BB subjects. Also, monocytes and macrophages from Nramp1AA subjects displayed a higher number of viable intracellular bacteria in comparison with monocytes and macrophages from Nramp1BB animals, providing biological significance to the results from association studies.

Puroindolines are antimicrobial peptides that occur in wheat seed, and are characterized by broad antimicrobial activity. We describe the heterologous expression of puroindoline A and B in the Origami strain of Escherichia coli . The recombinant puroindolines showed the same antimicrobial activity on Staphylococcus epidermidis as compared to the native peptides (MIC 90 = 30 μg ml −1 ). The bactericidal activity was 125 μg ml −1 for recombinant puroindoline A and 42 μg ml −1 for recombinant puroindoline B. Neither protein shows in vitro haemolytic activity or toxicity towards the murine macrophage cell line J774, but they are able to kill intracellular staphylococci. Our preliminary results suggest that recombinant puroindolines deserve further attention as alternatives to the conventional antibiotics in the control of S. epidermidis skin infections.

A bacteriophage lytic for Escherichia coli O157:H7 was isolated from bovine manure. Following in-vivo selection, the phage acquired the capacity to persist in the circulatory system of mice for at least 38 days. When mice were infected experimentally with E. coli O157:H7 (107 CFU/mouse), simultaneous injection of the mice with phage (108 PFU/mouse) cleared E. coli O157:H7 from the mice within 48 h.

Puroindolines, for years largely investigated for their involvement in wheat kernel hardness, have recently attracted attention thanks to their possible role as antimicrobial proteins. With the aim to enhance our knowledge of these proteins we studied their localization in the kernel, and their antimicrobial activity in vitro against six different bacterial strains. Immunolocalization showed that both the PINs are strongly concentrated in the aleurone layer, but also highly present in the endosperm. Interestingly we observed that puroindolines not only have the same spatial distribution in the kernel, they are also always found co-localized. Their co-localization suggests that they could cooperate in defending the plant against pathogens. We therefore tested antimicrobial activity of PINA and PINB, and a putative synergism between these proteins. The results showed that the two polypeptides can in vitro inhibit growth of all the bacteria tested; furthermore when combined together they are able to enhance each other's toxicity. In view of their antimicrobial activity and of their natural presence in Triticum aestivum wheat flour, puroindolines look promising antibacterial agents and thus deserve further studies aimed at establishing their possible future applications in fields of food and health care. Since PINs were still detectable in bakery products, these proteins may be promising tools in investigating natural ways of food preservation.

The genetic relationship among commercial cultivars of Citrus limon (lemon) was analysed by inter-simple sequence repeats (ISSR) and flow cytometry techniques. Two cultivars with a close germplasm were distinguished by screening 10 SSR primers and by measuring DNA content of prestained nuclei.

Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage phi31 and good acid production. Strain Str31 is very sensitive to phage phi31. This phage-host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study. Mutants were stable with respect to both characters. The isolation of the double mutants required 4 to 5 days. The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied.

Background Antimicrobial peptides (AMPs) are an ancient group of defense molecules. AMPs are widely distributed in nature (being present in mammals, birds, amphibians, insects, plants, and microorganisms). They display bactericidal as well as immunomodulatory properties. The aim of this study was to investigate the antimicrobial and anti-inflammatory activities of a combination of two AMPs (temporin B and the royal jellein I) against Staphylococcus epidermidis. Results The temporin B (TB-KK) and the royal jelleins I, II, III chemically modified at the C terminal (RJI-C, RJII-C, RJIII-C), were tested for their activity against 10 different Staphylococcus epidermidis strains, alone and in combination. Of the three royal jelleins, RJI-C showed the highest activity. Moreover, the combination of RJI-C and TB-KK (MIX) displayed synergistic activity. In vitro, the MIX displayed low hemolytic activity, no NO2- production and the ability to curb the synthesis of the pro-inflammatory cytokines TNF-α and IFN-γ to the same extent as acetylsalicylic acid. In vivo, the MIX sterilized mice infected with Staphylococcus epidermidis in eleven days and inhibited the expression of genes encoding the prostaglandin-endoperoxide synthase 2 (COX-2) and CD64, two important parameters of inflammation. Conclusion The study shows that the MIX – a combination of two naturally occurring peptides - displays both antimicrobial and anti-inflammatory activities.

A monoclonal antibody against goat immunoglobulins recognizes an allotypic determinant (A1) which is common to goat, sheep, cattle and water buffalo. The frequency of the corresponding gene (A') is about the same in all four species, indicating the existence of a polymorphism that remained stable over a period of about 18-20 million years.

Four formae speciales of F. oxysporum (dianthi, melonis, pisi, lycopersici) and three physiological races (1, 2, 3) of F. oxysporum f.sp. melonis have been differentiated using specific antisera and the techniques of double diffusion and radio double diffusion in agar. Three isolates of F. oxysporum f.sp. dianthi race 2 and 10 of f.sp. lycopersici race 1 tested against the appropriate antisera reacted the same within each race. The classification of fungi causing disease in plants is at present primarily based on morphological criteria and in the great majority of cases, these prove adequate. However, when the need arises to differentiate between formae speciales of the same species or even physiological races of the sameforma specialis, pathogenicity tests become necessary. Within the genus Fusarium Link ex Fr., whose taxonomy is as yet controversial (Wollenweber and Reinking, 1935; Snyder and Hansen, 1940, 1941, 1945; Bilai, 1955; Snyder et al., 1957; Booth, 1971), the species Fusarium oxysporum was chosen because of its distribution and importance. However, the identification of the 76 formae speciales and the very numerous physiological races of F. oxysporum Schlecht. emend. Snyd. et Hans. (Booth, 1971) by pathogenicity tests alone is a difficult and time-consuming task for the pathologist. Quick and reliable tests, either alternative or complementary to those based on pathogenicity, have therefore been sought (Tempel, 1959; Hornok, 1980; Marziano et al., 1981). In this paper we show that F. oxysporum, F. moniliforme and F. xylarioides possess distinct antigenic characteristics. In addition, we describe how four differentformae speciales of F. oxysporum (dianthi, melonis, pisi, lycopersici) and three physiological races of F. oxysporum f.sp. melonis (races 1, 2, 3) can be differentiated by serological techniques.

The old Annurca apple cultivar (Malus domestica), particularly appreciated for its peculiar flavor and crispy flesh, was studied in order to preserve its ancient germplasm. Twelve clones of Annurca were analyzed using random amplified polymorphic DNA (RAPD) and simple-sequence repeat (SSR) markers. Two out of 30 RAPD primers and nine out of ten SSR primers were able to discriminate all the clones analyzed. Data were confirmed by measuring DNA content using flow cytometry. The results provide a good procedure to improve germplasm field management, in order to removing redundant material in the Annurca collection. This represents an efficient way to create a data bank in order to preserve the genetic variability of the Annurca cultivar.