Molecular battles between plant and pathogenic bacteria in the phyllosphere

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.

The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

Non-biting flying insects as carriers of pathogenic bacteria in a Brazilian hospital

IntroductionInsects have been described as mechanical vectors of nosocomial infections.MethodsNon-biting flying insects were collected inside a pediatric ward and neonatal-intensive care unit (ICU) of a Brazilian tertiary hospital.ResultsMost (86.4%) of them were found to carry one or more species of bacteria on their external surfaces. The bacteria isolated were Gram-positive bacilli (68.2%) or cocci (40.9%), and Gram-negative bacilli (18.2%).ConclusionsInsects collected inside a hospital were carrying pathogenic bacteria; therefore, one must consider the possibility they may act as mechanical vectors of infections, in especially for debilitated or immune-compromised patients in the hospital environments where the insects were collected.

Synergistic interactions in mixed-species biofilms of pathogenic bacteria from the respiratory tract

IntroductionMixed-species biofilms are involved in a wide variety of infections. We studied the synergistic interactions during dual-species biofilm formation among isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia.MethodsIsolates were cultured as single-species and all possible combinations of dual-species biofilms.ResultsThe 61 A. baumannii biofilms increased by 26-fold when cultured with S. maltophilia isolates; 62 A. baumannii biofilms increased by 20-fold when cultured with S. maltophilia isolates; and 31 P. aeruginosa biofilms increased by 102-fold when cultured with S. maltophilia 106.ConclusionsSynergy was observed between two isolates, including those that inherently lacked biofilm formation ability.

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.

Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria

A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 µg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 µg/mL but the lectin (10-1000 µg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 µg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 µg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

Synergistic and antimicrobial properties of commercial turmeric (Curcuma longa) essential oil against pathogenic bacteria

Several studies have shown the antimicrobial and antioxidant properties of turmeric (Curcuma longa), widely used in food industry as a colorant, among other functions. The aim of this study was to determine the antioxidant and antimicrobial properties of turmeric essential oil against pathogenic bacteria and to study the influence of the addition of ascorbic acid on the prevention of polyphenols oxidation. The commercial turmeric essential oil alone did not show bactericidal activity against the microorganisms studied, Listeria monocytogenes and Salmonella typhimurium, but when combined with ascorbic acid, it showed significant antibacterial activity. The highest antimicrobial activity of turmeric essential oil against Salmonella typhimurium was 15.0 ± 1.41 mm at the concentration of 2.30 mg.mL-1 of essential oil and 2.0 mg.mL-1 of ascorbic acid. With regard to Listeria monocytogenes, the largest zone of inhibition (13.7 ± 0.58 mm) was obtained at the same concentrations. The essential oil showed antioxidant activity of EC50 = 2094.172 µg.mL-1 for the DPPH radical scavenging method and 29% under the concentration of 1.667 mg.mL-1 for the β-carotene bleaching method.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.). Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

Antibacterial and antifungal properties of wax and hexane extracts of Citrus spp. peels were tested using bioautographic and microdilution techniques against three plant pathogenic fungi (Penicillium digitatum, Curvularia sp., and Colletotrichum sp.), two human pathogens (Trichophyton mentagrophytes and Microsporum canis), and two opportunistic bacteria (Escherichia coli and Staphylococcus aureus). Two polymethoxylated flavonoids and a coumarin derivative, were isolated and identified from peel extracts, which presented antimicrobial activity especially against M. canis and T. mentagrophytes: 4',5,6,7,8-pentamethoxyflavone (tangeritin) and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin) from C. reticulata; and 6,7-dimethoxycoumarin (also known as escoparone, scoparone or scoparin) from C. limon.

Antibacterial activity of essential oils on Xanthomonas vesicatoria and control of bacterial spot in tomato

The objective of this work was to evaluate the effects of plant essential oils (EOs) on the growth of Xanthomonas vesicatoria, on bacterial morphology and ultrastructure, and on the severity of tomato bacterial spot. EOs from citronella, clove, cinnamon, lemongrass, eucalyptus, thyme, and tea tree were evaluated in vitro at concentrations of 0.1, 1.0, 10, and 100% in 1.0% powdered milk. The effect of EOs, at 0.1%, on the severity of tomato bacterial spot was evaluated in tomato seedlings under greenhouse conditions. The effects of citronella, lemongrass, clove, and tea tree EOs, at 0.1%, on X. vesicatoria cells were evaluated by transmission electron microscopy. All EOs showed direct toxic effect on the bacteria at a 10%-concentration in vitro. Under greenhouse conditions, the EOs of clove, citronella, tea tree, and lemongrass reduced disease severity. EOs of clove and tea tree, and streptomycin sulfate promoted loss of electron-dense material and alterations in the cytoplasm, whereas EO of tea tree promoted cytoplasm vacuolation, and those of citronella, lemongrass, clove, and tea tree caused damage to the bacterial cell wall. The EOs at a concentration of 0.1% reduce the severity of the disease.

Recent research data on the removal of pharmaceuticals from sewage treatment plants (STP)

Pharmaceutical compounds have been detected in sewage treatment plant (STP) effluents, surface waters and, less frequently, in groundwater and drinking water, all over the world. Different sources are responsible for their appearance in the aquatic environment, however, it is widely accepted that the main sources of this type of pollutant are STP effluents. The adverse effects of pharmaceuticals in the environment include aquatic toxicity, development of resistance in pathogenic bacteria, genotoxicity and endocrine disruption. Thus, the discharge of these compounds to the environment in STP effluents should be minimized.

Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.