Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC₅₀ and IC₉₀, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC₅₀ > 4.7 μM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC₅₀ < 0.2 μM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC₅₀ between 0.4 and 0.9 μM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC₅₀ between 1.0 and 1.2 μM). In cluster 4 were species with moderate to high tolerance (IC₅₀ between 1.3 and 1.6 μM). Cluster 5 contained the species with high tolerance (IC₅₀ between 1.9 and 4.0 μM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.     

Oxacillinases and antimicrobial susceptibility of Ralstonia pickettii from pharmaceutical water systems in Croatia

This study evaluated antibiotic susceptibility and presence of bla_OXA22 and bla_OXA60 genes in 81 isolates of Ralstonia pickettii obtained from different purified and ultra-pure water systems in two different geographical areas of Croatia. E-test and disc diffusion test were performed to determine antibiotic susceptibility. Polymerase chain reaction was applied to detect genes encoding OXA-22 and OXA-60 oxacillinases previously identified in R. pickettii. The isolates were genotyped by pulsed-field gel electrophoresis. The results revealed variable susceptibility/resistance profiles. Our isolates exhibited high susceptibility rates to ceftriaxone, cefotaxime, piperacillin-tazobactam, ciprofloxacin, imipenem, cefepime and in lesser extent to ceftazidime. High rates of susceptibility were also observed for sulphamethoxazole-trimethoprim and piperacillin. High resistance rates were noticed for ticarcillin-clavulanate, aztreonam and meropenem, as well as for all aminoglycosides tested. Modified Hodge test was positive in 51·9% strains, indicating production of carbapenemases. bla_OXA22 and bla_OXA60 genes were detected in 37·0 and 80·3% strains, respectively. Pulsed-field gel electrophoresis identified three major clusters containing subclusters. R. pickettii should be taken seriously as a possible cause of nosocomial infections to ensure adequate therapy, to prevent the development of resistant strains and to try to reduce the possibility of R. pickettii surviving in clean and ultra clean water systems.     

Comparison of Antibiotic Resistance, Biofilm Formation and Conjugative Transfer of Staphylococcus and Enterococcus Isolates from International Space Station and Antarctic Research Station Concordia

The International Space Station (ISS) and the Antarctic Research Station Concordia are confined and isolated habitats in extreme and hostile environments. The human and habitat microflora can alter due to the special environmental conditions resulting in microbial contamination and health risk for the crew. In this study, 29 isolates from the ISS and 55 from the Antarctic Research Station Concordia belonging to the genera Staphylococcus and Enterococcus were investigated. Resistance to one or more antibiotics was detected in 75.8 % of the ISS and in 43.6 % of the Concordia strains. The corresponding resistance genes were identified by polymerase chain reaction in 86 % of the resistant ISS strains and in 18.2 % of the resistant Concordia strains. Plasmids are present in 86.2 % of the ISS and in 78.2 % of the Concordia strains. Eight Enterococcus faecalis strains (ISS) harbor plasmids of about 130 kb. Relaxase and/or transfer genes encoded on plasmids from gram-positive bacteria like pIP501, pRE25, pSK41, pGO1 and pT181 were detected in 86.2 % of the ISS and in 52.7 % of the Concordia strains. Most pSK41-homologous transfer genes were detected in ISS isolates belonging to coagulase-negative staphylococci. We demonstrated through mating experiments that Staphylococcus haemolyticus F2 (ISS) and the Concordia strain Staphylococcus hominis subsp. hominis G2 can transfer resistance genes to E. faecalis and Staphylococcus aureus, respectively. Biofilm formation was observed in 83 % of the ISS and in 92.7 % of the Concordia strains. In conclusion, the ISS isolates were shown to encode more resistance genes and possess a higher gene transfer capacity due to the presence of three vir signature genes, virB1, virB4 and virD4 than the Concordia isolates.     

Diversity of moxifloxacin resistance during a nosocomial outbreak of a predominantly ribotype ARU 027 Clostridium difficile diarrhea

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB. All the laboratory isolates, including the historical ARU 027 isolate Cd196, were susceptible to moxifloxacin (≤2 μg/mL). 13 field isolates were susceptible to ≤2 μg/mL. Five were resistant to from 4 to 12 μg/mL (moderate resistance); 16 were resistant to ≥16 μg/mL (high resistance). We sequenced the quinolone resistance determining regions of gyrA (position 71-460) and gyrB (position 1059-1448) from two susceptible laboratory strains, all five isolates with moderate resistance and two highly resistant isolates. Two highly resistant isolates (Pitt 40, ribotype ARU 027 and Pitt 33, ribotype ARU 001) had the same C245T (Thr₈₂ΔIle) mutation. No other changes were seen. Amplification with primer pairs specific for the C245T mutant gyrA and for the wild type gene respectively confirmed all 16 highly resistant ARU 027 isolates, as well as the highly resistant isolates from other ribotypes, had the C245T mutation and that the mutation was absent from all other isolates. Among the five isolates with moderate resistance we found combinations of mutations within gyrA (T128A, Val₄₃ΔAsp and G349T, Ala₁₁₇ΔSer) and gyrB (G1276A, Arg₄₂₆ΔAsn). The G1396A (Glu₄₆₆ΔLys) mutation was not associated with increased resistance.     

<Emphasis Type="Italic">Ralstonia pickettii</Emphasis> Bloodstream Infections at a Brazilian Cancer Institution

We describe a series of Ralstonia pickettii bloodstream infections (BSI) that occurred in 19 oncohematologic patients admitted to a hospital for patients with cancer, in the city of Rio de Janeiro, from July 1999 to February 2006. Fifty-four R. pickettii isolates were recovered from blood and catheter-tip specimens (1–5 isolates per patient). Two patients eventually died of causes unrelated to R. pickettii BSI. Eight pulsed-field gel electrophoresis genotypes were resolved (A–H), with two detected in more than 1 patient: genotype B, in 2 patients (1.5%), and E, in 12 patients (63.2%). R. pickettii emerged as a new pathogen at our institution, causing at least one outbreak. Cross-transmission of the pathogen, infusion of a putative contaminated intravenous solution, and persistent colonization of medical devices were the likely sources of R. pickettii BSI.     

Specific and Sensitive Detection of Ralstonia solanacearum in Soil on the Basis of PCR Amplification of fliC Fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10³ CFU g of bulk soil⁻¹. The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Isolation and characterization of bacteria from mercury contaminated sites in Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability of a Pseudomonas putida V1 strain

Methylmercury (MeHg) is one of the most dangerous heavy metal for living organisms that may be found in environment. Given the crescent industrialization of Brazil and considering that mercury is a residue of several industrial processes, there is an increasing need to encounter and develop remediation approaches of mercury contaminated sites. The aim of this study was to isolate and characterize methylmercury resistant bacteria from soils and sludge sewage from Rio Grande do Sul, Brazil. Sixteen bacteria were isolated from these contaminated sites and some isolates were highly resistant to methylmercury (>8.7 μM). All the isolates were identified by 16S rDNA. Pseudomonas putida V1 was able to volatilize approximately 90 % of methylmercury added to growth media and to resist to copper, lead, nickel, chromate, zinc, cobalt, manganese and barium. In the presence of high concentrations of methylmercury (12 μM), cell growth was limited, but P. putida V1 was still able to remove up to 29 % of this compound from culture medium. This bacterium removed an average of 77 % of methylmercury from culture medium with pH in the range 4.0–6.0. In addition, methylmercury was efficiently removed (>80 %) in temperature of 21–25 °C. Polymerase chain reactions indicated the presence of merA but not merB in P. putida V1. The growth and ability of P. putida V1 to remove methylmercury in a wide range of pH (4.0 and 8.0) and temperature (10–35 °C), its tolerance to other heavy metals and ability to grow in the presence of up to 11.5 μM of methylmercury, suggest this strain as a new potential resource for degrading methylmercury contaminated sites.     

Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01

Biomineralization-inspired preparation of nanoparticles by marine microorganisms is in the limelight of modern nanotechnology. In recent years, the use of marine microorganisms for the synthesis of nanoparticles has been gaining importance due to the simplicity and eco-friendliness of the approach. Here we describe the synthesis of silver nanoparticles using halotolerant Bacillus sp. isolated from the southern coastal waters of India. Our selective and enriched isolation technique resulted in the isolation of a silver nitrate-resistant novel marine Bacillus sp. isolated from sediments collected at Ennore Port, Chennai, India. The strain was characterized by the polyphasic taxonomic approach, and phenotypic and phylogenetic analysis identified the strain as Bacillus sp. VITSSN01. The resistant strain was further assayed for the synthesis of silver nanoparticles and its biological activity evaluated. Nanoparticles were synthesized under optimized nutritional and cultural conditions with shaking and the production continuously monitored. The nanoparticles thus produced were then characterized by atomic force microscopy, X-ray diffraction, Fourier transform-infrared spectrophotometer and transmission electron microscopy. The mean particle size was 46 nm. Hemotological toxicity of nanoparticles is very severe form and less studied. We therefore checked the synthesized silver nanoparticles for toxicity against erythrocytes and found that the silver nanoparticles exhibited moderate hemolytic activity against human erythrocytes, with a half maximal effective concentration (EC₅₀) value of 60 μg/ml. Microscopic studies of the treated erythrocytes showed slight structural perturbations. The results of our study strongly suggest that marine microorganisms could be a potential source for the rapid and eco-friendly synthesis of nanoparticles.     

Molecular epidemiology of Clostridium difficile strains from nosocomial-acquired infections

The purpose of this study is to analyze isolates of Clostridium difficile from patients with nosocomial acquired infection in respect to their molecular type and antimicrobial susceptibility. Fifty-nine randomly selected clinical isolates were characterized. Molecular typing was performed by rep-PCR (DiversiLab). Isolates were tested by disk diffusion towards 11 different antibiotics. All isolates were susceptible to metronidazole and vancomycin. Fifty five (93 %) isolates were resistant to erythromycin and fifty six (95 %) exhibited resistance to both clindamycin and moxifloxacin. Twenty rep-PCR types were identified, but most clinical isolates formed four major rep-PCR clusters (A₁ 24/59, 40 %; A₂ 20/59, 33 %; A₃ 5/59, 8 %; A₄ 3/59, 5 %). These results show high genetic variability, which demonstrate clearly the complexity of the strains of C. difficile and also show an increasing rate of resistance to fluoroquinolones in our region emphasizing the importance of implementing surveillance programs in order to prevent further spread of resistance in C. difficile.     

Veronaea botryosa: Molecular Identification with Amplified Fragment Length Polymorphism (AFLP) and In vitro Antifungal Susceptibility

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC₉₀s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).     

Mycotoxigenic fungi and mycotoxins associated with stored maize from different regions of Lesotho

Samples of stored maize from villages located in five different agroecological zones (southern lowlands, northern lowlands, Senqu river valley, foothills and mountains) of Lesotho were collected in 2009/10 and 2010/11 and assessed for contamination with toxigenic fungi. The water activity of all samples collected during the two seasons was <0.70. The total fungal populations of the maize from different regions in the two seasons was not significantly different (p?>?0.05). Fusarium verticillioides, F. proliferatum and F. subglutinans predominated in different regions in both seasons based on molecular analyses. In the 2009/10 season, the isolates of these species all produced FB₁, while in the 2010/11 season, very few produced FB₁. A. flavus isolates (2009/10) were recovered from mountains and Senqu river valley samples while the 2010/11 isolates were predominantly from the foothills and northern lowlands. The mountain isolates of Aspergillus section Flavi produced the highest levels of AFB₁ (20 mg kg^?1). Aspergillus parasiticus was only isolated from the foothills, Senqu river valley and southern lowlands samples, and the AFB₁ levels produced ranged from ‘none detected’ to 3.5 mg kg^?1. The Aspergillus ochraceous isolates were least frequently encountered in both seasons. In the 2009/10 season, the isolates from the northern lowlands produced ochratoxin A (OTA) in culture. No isolates of A. niger from different regions in both seasons produced any OTA. Multi-mycotoxin analyses of the maize samples were done for a range of mycotoxins. At least one sample from each region in both seasons was FB₁-positive. FB₁ levels for 2010/11 samples (7–936 μg kg^?1) were higher than in the 2009/10 season (2–3 μg kg^?1). In both seasons, the mountains registered the highest levels of FB₁. Deoxynivalenol (DON) was recovered from all the samples analysed, with the highest mean contamination of 1,469 μg kg^?1 in samples from the northern lowlands. Moniliformin (MON) was detected from all agroecological zones in the two seasons (5–320 μg kg^?1 in 2009/10; 15–1,205 μg kg^?1 in 2010/11). Emerging toxins such as fusaproliferin (FUS) and beauvericin (BEA) were also detected. OTA was not detected in any of the samples analysed. Only one 2009/10 sample in the Senqu river valley was positive for AFB₁. This is the first report on toxigenic fungi and multi-mycotoxin contamination of maize samples from subsistence farmers’ stores in different agroecological zones of Lesotho.     

Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2 % in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1–2 mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1 mm). Majority of R. solani isolates showed <8 μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6 %) of AG3 was the highest followed by AG2–3 (20.9 %) and AG5 (17.4 %). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3 %). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.     

Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments

Suaeda fruticosa Forssk is a leaf succulent obligate halophyte that produces numerous seeds under saline conditions. Seeds are a good source of high quality edible oil and leaves are capable of removing substantial amount of salt from the saline soil besides many other economic usages. Little is known about the biochemical basis of salt tolerance in this species. We studied some biochemical responses of S. fruticosa to different exogenous treatments under non-saline (0 mM), moderate (300 mM) or high (600 mM) NaCl levels. Eight-week-old seedlings were sprayed twice a week with distilled water, hydrogen peroxide (H₂O₂, 100 μM), glycine betaine (GB, 10 mM), or ascorbic acid (AsA, 20 mM) for 30 days. At moderate (300 mM) NaCl, leaf Na⁺, Ca²⁺ and osmolality increased, along with unchanged ROS and antioxidant enzyme activities, possibly causing a better plant growth. Plants grew slowly at 600 mM NaCl to avoid leaf Na⁺ buildup relative to those at 300 mM NaCl. Exogenous application of distilled water and H₂O₂ improved ROS scavenging mechanisms, although growth was unaffected. ASA and GB alleviated salt-induced growth inhibition at 600 mM NaCl through enhancing the antioxidant defense system and osmotic and ion homeostasis, respectively.     

Phosphate solubilization potential and modeling of stress tolerance of rhizobacteria from rice paddy soil in northern Iran

The purposes of this study were to evaluate the phosphate solubilization activity of bacteria isolated from the rhizosphere of rice paddy soil in northern Iran, and to study the effect of temperature, NaCl and pH on the growth of these isolates by modeling. Three of the most effective strains from a total of 300 isolates were identified and a phylogenetic analysis was carried out by 16S rDNA sequencing. The isolates were identified as Pantoea ananatis (M36), Rahnella aquatilis (M100) and Enterobacter sp. (M183). These isolates showed multiple plant growth-promoting attributes such as phosphate solubilization activity and indole-3-acetic acid (IAA) production. The M36, M100 and M183 isolates were able to solubilize 172, 263 and 254 µg ml^?1 of Ca₃(PO₄)₂ after 5 days of growth at 28 °C and pH 7.5, and to produce 8.0, 2.0 and 3.0 μg ml^?1 of IAA when supplemented with l-tryptophan (1 mg ml^?1) for 72 h, at 28 °C and pH 7.0, respectively. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium and there was an inverse relationship between pH and solubilized P (r = ?0.98, P < 0.0952). There were no significant differences among isolates in terms of acidity tolerance based on their confidence limits as assessed by segmented model analysis and all isolates were able to grow at pH 4.3–11 (with optimum at 7.0–7.5). Based on a sigmoidal trend of a three-parameter logistic model, the salt concentration required for 50 % inhibition was 8.15, 6.30 and 8.23 % NaCl for M36, M100 and M183 isolates, respectively. Moreover, the minimum and maximum growth temperatures estimated by the segmented model were 5.0 and 42.75 °C for M36, 12.76 and 40.32 °C for M100, and 10.63 and 43.66 °C for M183. The three selected isolates could be deployed as inoculants to promote plant growth in an agricultural environment.     

Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis

Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92 %, 34/37) and penicillin (89 %, 33/37) followed by resistance towards ampicillin (68 %, 25/37), cefuroxime (38 %, 14/37), amikacin (6 %, 2/37) and ceftazidime (14 %, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80 %. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus.     

The regulatory role of riboflavin in the drought tolerance of tobacco plants depends on ROS production

Riboflavin (vitamin B₂) is required for normal plant growth and development. Previous studies have shown that riboflavin application can enhance pathogen resistance in plants. Here, we investigated the role of riboflavin in increasing drought tolerance (10 % PEG6000 treatment) in plants. We treated 4 week-old tobacco plants with five different levels of riboflavin (0, 4, 20, 100 and 500 μM) for 5 days and examined their antioxidant responses and levels of drought tolerance. Compared with the controls, low and moderate levels of riboflavin treatment enhanced drought tolerance in the tobacco plants, whereas higher concentrations of riboflavin (500 μM) impaired drought tolerance. Further analysis revealed that plants treated with 500 μM riboflavin accumulated higher levels of ROS (O₂ ^? and H₂O₂) and lipid peroxide than the control plants or plants treated with low levels of riboflavin. Consistent with this observation, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were higher in plants treated with low or moderate (4, 20 and 100 μM) levels of riboflavin compared with the control. We also found that chlorophyll degraded rapidly in control and 500 μM riboflavin-treated plants under drought stress conditions. In addition, the survival times of the riboflavin-treated plants were significantly modified by treatment with reduced glutathione, a well-known ROS scavenger, under drought stress conditions. Thus, riboflavin-mediated ROS production may determine the effects of riboflavin on drought tolerance in tobacco plants.     

In Vitro Activity of New Cephalosporins vs Streptococcus pneumoniae from the Canadian Bacterial Surveillance Network: 2008–2011

Between 2008 and 2011, 6,895 Streptococcus pneumoniae isolates were submitted to the Canadian Bacterial Surveillance Network and underwent in vitro susceptibility testing. Fifteen percent of S. pneumoniae isolates were collected from pediatric patients (0–15 years old), 48.6 % of isolates were collected from adults between 16 and 64 years of age, and 36.1 % from adults aged ≥65 years; age data were not available for 11 patients. Forty-five percent of S. pneumoniae isolates were recovered from sterile specimens, and 55 % of isolates were from nonsterile specimens. Overall, 0.4 % of isolates were resistant to penicillin, 0.4 % to ceftriaxone, 3 % to amoxicillin, 25 % to erythromycin, and 13 % to trimethoprim/sulfamethoxazole; 6.6 % of isolates were multidrug resistant (MDR). Among MDR isolates, resistance rates exceeded 95 % for erythromycin, tetracycline, and trimethoprim/sulfamethoxazole. The MIC₉₀ of cethromycin, ceftaroline, and ceftobiprole against MDR isolates were 0.12, 0.25, and 1 mg/L, respectively. Ceftaroline, the active form of the prodrug ceftaroline fosamil, exhibited potent in vitro activity against the tested S. pneumoniae including all 456 multidrug-resistant strains. No ceftaroline-resistant isolates were identified.     

Chromosomal Sil system contributes to silver resistance in <Emphasis Type="Italic">E. coli</Emphasis> ATCC 8739

The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO₃. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO₃R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO₃R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.     

Drug Resistance of Enteric Bacteria IX. Distribution of R Factors in Gram-negative Bacteria from Clinical Sources

Many isolates belonging to the Enterobacteriaceae were collected in 1965 from the inpatients at geographically scattered hospitals in Japan. Among 2,650 Shigella strains examined, 58.4% were found to be drug-resistant; 95.0% of these resistant strains were multiply resistant. Among 434 resistant strains examined, 81% carried R factors that were transferable by cell-to-cell contact. Of 160 isolates of other enteric bacteria, drug-resistant strains included 84.2% of the Escherichia coli, 93.0% of the Klebsiella, and 90.0% of the Proteus cultures. Among these resistant strains, 70.3% of the E. coli, 66.7% of the Klebsiella, and 52.0% of the Proteus were multiply resistant. Of these resistant strains, 84.0% of the E. coli, 88.0% of the Klebsiella, and 50.0% of the Proteus strains carried R factors. These results indicate that R factors are widespread among gram-negative bacteria of clinical significance.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.