Volume 66 (2017): Issue 2 (January 2017)

Biochar is a solid material of biological origin obtained from biomass carbonization, designed as a mean to reduce greenhouse gases emission and carbon sequestration in soils for a long time. Biochar has a wide spectrum of practical utilization and is applied as a promising soil improver or fertilizer in agriculture, or as a medium for soil or water remediation. Preparations of biochar increase plant growth and yielding when applied into soil and also improve plant growth conditions, mainly bio, physical and chemical properties of soil. Its physical and chemical properties have an influence on bacteria, fungi and invertebrates, both in field and laboratory conditions. Such effects on rhizosphere organisms are positive or negative depending on biochar raw material origin, charring conditions, frequency of applications, applications method and doses, but long term effects are generally positive and are associated mainly with increased soil biota activity. However, a risk assessment of biochar applications is necessary to protect food production and the soil environment. This should be accomplished by biochar production and characterization, land use implementation, economic analysis, including life cycle assessment, and environmental impact assessment.

Aminoglycosides are used in treating a wide range of infections caused by Gram-positive and Gram-negative bacteria; however, aminoglycoside resistance is common and occurs by several mechanisms. Among these mechanisms is bacterial rRNA methylation by the 16S rRNA methyl transferase (16S-RMTase) enzymes; but data about the spread of this mechanism in Egypt are scarce. Cephalosporins are the most commonly used antimicrobial agents in Egypt; therefore, this study was conducted to determine the frequency of 16S-RMTase among third generation cephalosporin-resistant clinical isolates in Egypt. One hundred and twenty three cephalosporin resistant Gram-negative clinical isolates were screened for aminoglycosides resistance by the Kirby Bauer disk diffusion method and tested for possible production of 16S-RMTase. PCR testing and sequencing were used to confirm the presence of 16S-RMTase and the associated antimicrobial resistance determinants, as well as the genetic region surrounding the armA gene. Out of 123 isolates, 66 (53.66%) were resistant to at least one aminoglycoside antibiotic. Only one Escherichia coli isolate (E9ECMO) which was totally resistant to all tested aminoglycosides, was confirmed to have the armA gene in association with bla_TEM-1, bla_CTX-M-15, bla_CTX-M-14 and aac(6)-Ib genes. The armA gene was found to be carried on a large A/C plasmid. Genetic mapping of the armA surrounding region revealed, for the first time, the association of armA with aac(6)-Ib on the same transposon. In conclusion, the isolation frequency of 16S-RMTase was low among the tested aminoglycosideresistant clinical samples. However, a novel composite transposon has been detected conferring high-level aminoglycosides resistance.

The aim of the study was to assess the epidemiology, the incidence of multidrug-resistant bacteria and bloodstream infections’ (BSIs) seasonality in a university hospital. This retrospective study was carried out in the University General Hospital of Patras, Greece, during 2011–13 y. Blood cultures from patients with clinical presentation suggestive of bloodstream infection were performed by the BacT/ALERT System. Isolates were identified by Vitek 2 Advanced Expert System. Antibiotic susceptibility testing was performed by the disk diffusion method and E-test. Resistance genes (mecA in staphylococci; vanA/vanB/vanC in enterococci; bla_KPC/bla_VIM/bla_NDM in Klebsiella spp.) were detected by PCR. In total, 4607 (9.7%) blood cultures were positive from 47451 sets sent to Department of Microbiology, representing 1732 BSIs. Gram-negative bacteria (52.3%) were the most commonly isolated, followed by Gram-positive (39.5%), fungi (6.6%) and anaerobes bacteria (1.8%). The highest contamination rate was observed among Gram-positive bacteria (42.3%). Among 330 CNS and 150 Staphylococcus aureus, 281 (85.2%) and 60 (40.0%) were mecA-positive, respectively. From 113 enterococci, eight were vanA, two vanB and two vanC-positives. Of the total 207 carbapenem-resistant Klebsiella pneumoniae (73.4%), 202 carried bla_KPC, four bla_KPC and bla_VIM and one bla_VIM. A significant increase in monthly BSIs’ incidence was shown (R²: 0.449), which may be attributed to a rise of Gram-positive BSIs (R²: 0.337). Gram-positive BSIs were less frequent in spring (P < 0.001), summer (P < 0.001), and autumn (P < 0.001), as compared to winter months, while Gram-negative bacteria (P < 0.001) and fungi (P < 0.001) were more frequent in summer months. BSIs due to methicillin resistant S. aureus and carbapenem-resistant Gram-negative bacteria increased during the study period. The increasing incidence of BSIs can be attributed to an increase of Gram-positive BSI incidence, even though Gram-negative bacteria remained the predominant ones. Seasonality may play a role in the predominance of Gram-negative’s BSI.

Growth kinetics of four Trichoderma strains was tested on lignocellulosic by-products in solid state fermentation (SSF). The strains were also analyzed for their survival rate and growth after lyophilization on these carriers. All applied monocomponent and bicomponent media were substrates for the production and preservation of Trichoderma biomass. However, the maximum number of colony forming units (CFU/g dm) was acquired on bicomponent media based on dried grass and beet pulp or grass with corn cobs, when compared to monocomponent media. Although the process of lyophilization reduced the survival rate by 50–60%, the actual number of viable cells in obtained biopreparations remained relatively high (0.58 × 10⁸ – 1.68 × 10⁸ CFU/g dm). The studied strains in the preserved biopreparations were characterized by a high growth rate, as evaluated in microcultures using the Bioscreen C system.

Pseudomonas putida strain (HM346961) was isolated from a consortium of bacteria acclimatized to unleaded gasoline-contaminated water. The consortium can efficiently remove benzene, toluene, ethylbenzene and xylene (BTEX) isomers, and a similar capability was observed with the P. putida strain. Proteome of this strain showed certain similarities with that of other strains exposed to the hydrocarbon compounds. Furthermore, the toluene di-oxygenase (tod) gene was up-regulated in P. putida strain when exposed to toluene, ethylbenzene, xylene, and BTEX. In contrast, the tod gene of P. putida F1 (ATCC 700007) was up-regulated only in the presence of toluene and BTEX. Several differences in the nucleotide and protein sequences of these two tod genes were observed. This suggests that tod up-regulation in P. putida strain may partially explain their great capacity to remove aromatic compounds, relative to P. putida F1. Therefore, new tod and P. putida strain are promising for various environmental applications.

Agaricus bisporus plays an important role in ecological processes and is one of the most widely cultivated mushrooms worldwide. Mushroom growth-promoting bacteria have been isolated from casing soil and compost, but microorganisms in the fruiting body have received only a little attention. To get an overview of phylogenetic diversity of microorganisms in the fruiting body of A. bisporus, as well as to screen antimicrobial and mushroom growth-promoting strains, and eventually intensify mushroom production, we isolated and characterized microorganisms from the fruiting body of A. bisporus. In total, 55 bacterial strains were isolated, among which nine isolates represented Actinomycetes. All the isolates were analyzed by 16S rRNA gene RFLP and sixteen representative strains by 16S rRNA gene sequencing. According to the phylogenetic analysis, eleven isolates represented the Gram-positive Bacillus, Lysinibacillus, Paenibacillus, Pandorea and Streptomyces genera, and five isolates belonged to the Gram-negative Alcaligenes and Pseudomonas genera. The bacteria isolated from the fruiting body of A. bisporus had broad-spectrum antimicrobial activities and potential mushroom growth-promoting abilities.

Community structure of bacteria present in arsenic contaminated agricultural soil was studied with qPCR (quantitative PCR) and DGGE (Denaturing Gradient Gel Electrophoresis) as an indicator of extreme stresses. Copy number of six common bacterial taxa (Acidobacteria, Actinobacteria, α-, β- and γ-Proteobacteria, Firmicutes) was calculated using group specific primers of 16S rDNA. It revealed that soil contaminated with low concentration of arsenic was dominated by both Actinobacteria and Proteobacteria but a shift towards Proteo bacteria was observed with increasing arsenic concentration, and number of Actinobacteria eventually decreases. PCA (Principle Component Analysis) plot of bacterial community composition indicated a distinct resemblance among high arsenic content samples, while low arsenic content samples remained separated from others. Cluster analysis of soil parameters identifies three clusters, each of them was related to the arsenic content. Further, cluster analysis of 16S rDNA based DGGE fingerprint markedly distributed the soil bacterial populations into low (< 10 ppm) and high (> 10 ppm) arsenic content subgroups. Following analysis of diversity indices shows significant variation in bacterial community structure. MDS (Multi Dimensional Scaling) plot revealed distinction in the distribution of each sample denoting variation in bacterial diversity. Phylogenetic sequence analysis of fragments excised from DGGE gel revealed the presence of γ-Proteobacteria group across the study sites. Collectively, our experiments indicated that gradient of arsenic contamination affected the shape of the soil bacterial population by significant structural shift.

The purpose of this study was to determine the antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. isolated from different cheeses and also investigate some of their virulence factors. Enterococcus strains were isolated from 33 different cheeses. Enterococcus faecium (6 strains) and Enterococcus faecalis (5 strains) enterocin-producing strains were identified by 16S rDNA analyses. Structural genes entA, entB, entP and entX were detected in some isolates. Multiple enterocin structural genes were found in 7 strains. None of the tested enterococci demonstrated anyβ-haemolytic activity and only one strain had gelatinase activity. Six strains showed multiple antibiotic resistance patterns and in addition, vanA and several virulence genes were detected in many strains. Only E. faecalis MBE1-9 showed tyrosine decarboxylase activity and tdc gene was detected only in this strain.

An Egyptian, plant pathogenic Fusarium solani isolate was grown on cobalt concentrations of 0, 50, 200, 500, 800, and 1000 ppm. The isolate survived concentrations up to 800 ppm, however failed to grow at 1000 ppm. Morphology and elemental analysis of the isolate under the investigated Co concentrations were studied using Scanning electron microscopy (SEM) and energy dispersive X-ray microanalysis (EDX). The isolate reserved its morphology up to a concentration of 200 ppm. Morphological distortions were dramatic at 500 and 800 ppm. EDX detected Co uptake through the hyphae, microconidia, macroconidia, and chlamydospores. Iron, calcium, and potassium were also detected. EDX results showed a linear relationship between Co% and Fe% up to a concentration of 500 ppm reflecting the possible ability of the isolate to synthesize intracellular siderophores storing iron and their release from the vacuoles. The participation of such siderophores in conferring tolerance against cobalt is discussed. At 800 ppm, the % of Fe was greatly reduced with an accompanying increase in morphological distortions and absence of microconidia. Increasing the implicated cobalt concentrations resulted in increasing the percentages of the chelated cobalt reflecting the possible implication of the cell wall as well as extracellular siderophores in the uptake of cobalt. The current results recommend the absence of cobalt in any control regime taken to combat the investigated F. solani isolate and highlights the accomplishment of biochemical, ultrastructural, and molecular studies on such isolate to approve the production of siderophores and the role of cell wall in cobalt uptake.

The caecal chyme of pigs was incubated anaerobically in McDougall buffer with and without fumonisin B₁ (5 μg/ml) for 0, 24 and 48 h. The plate count agar technique was applied for enumerating the amount of bacteria including aerobic, anaerobic bacteria, coliform, Escherichia coli and Lactobacillus sp. The quantitative polymerase chain reaction was also performed to estimate the number of copies of the total bacteria, Lactobacillus, Bacteroides and Prevotella. No significant differences in the amount of bacterial groups between the experimental (buffer, chyme, and fumonisin B₁) and control 1 groups (buffer + chyme) were observed in both methods. Fumonisin B₁ and hydrolysed fumonisin B₁ concentration were analysed by liquid chromatograghy – mass spectrometry. There was no significant difference in FB₁ concentration between the experimental and the control 2 group (buffer and fumonisin B₁) at 0 h incubation, 5.185 ± 0.174 μg/ml compared with 6.433 ± 0.076 μg/ml. Fumonisin B₁ concentration in the experimental group was reduced to 4.080 ± 0.065 μg/ml at 24 h and to 2.747 ± 0.548 μg/ml at 48 h incubation and was significantly less than that of in the control group. Hydrolysed fumonisin B₁ was detected after 24 h incubation (0.012 ± 0 μg/ml). At 48 h incubation time, hydrolysed fumonisin B₁ concentration was doubled to 0.024 ± 0.004 μg/ml. These results indicate that fumonisin B₁ can be metabolised by caecal microbiota in pigs though the number of studied bacteria did not change.

Among 140 patients colonized by KPC-producing Klebsiella pneumoniae (KPC-Kp) between fourth and seventh day of Intensive Care Unit stay, 24 developed bacteraemia immediately after colonization. Colistin-resistance of the colonizing isolate was the factor significantly associated with early KPC-Kp bacteraemia (P < 0.001; OR 6.6, 95% CI 2.4–18.4), a worrisome finding since infections by colistin-resistant isolates is associated with increased mortality due to limited remaining therapeutic options.

Several studies have observed that use of a conventional PCR protocol with primers LM1 and LM2 for the identification of the hlyA gene of Listeria monocytogenes generates non-specific PCR amplifications and false positives. For this reason, in this study we provide a modified PCR protocol that improves the specificity of the results obtained with LM1 and LM2 primers.

The aim of our studies was to invent a reliable method for detection of the bactericidal activity of disinfectants against Borrelia burgdorferi in suspension (in vitro) and in cell line cultures (in vivo). In the suspension method, 0.01% octenidine at 20°C and 35°C was bactericidal to Borrelia afzeli; Borrelia garini, B. burgdorferi sensu stricto after 5 minutes treatment. Increase of the temperature to 35°C speed up the bactericidal effect to 1 minute. The bactericidal action of octenidine towards B. burgdorferi spirochetes growing in fibroblasts was less effective and needed a longer time to kill them than in the suspension.

Retrospective analysis of Streptococcus agalactiae antibiotic susceptibility isolated in 2010–2013 was performed. Penicillin was still the firstline antibiotic. Due to the high percentage of strains resistant to erythromycin and clindamycin empirical treatment with these antibiotics may not be effective. Lower resistance rate to erythromycin and clindamycin among strains isolated from infected pregnant women and newborns were observed than among strains isolated from samples from patients hospitalized in other departments (29% and 47% v. 46% and 63%). The increasing resistance rate might give a rise to a new epidemiological situation.

The aim of this study was the isolation of Pseudomonas sp. and Bacillus sp. strains from rhizospheric soil and monitoring the impact of two isolates denoted as P12 (Pseudomonas sp.) and B1 (Bacillus sp.) on the parameters of English ryegrass (Lolium perenne) yield and activity of the soil microbial communities. During 2012–2014, a plot experiment was set up following the randomized block system. Better effect on the plant growth was recorded with the use of Pseudomonas sp. P12 isolate than with Bacillus sp. B1. Positive effect on the increase in the total number of microorganisms, aminoheterotrophs and azotobacter was also achieved. Bacillus sp. B1 increased only the number of actimycetes. Both isolates positively affected dehydrogenase activity (DHA).

The traditional CaCO₃-based fermentation process generates huge amount of insoluble waste. To solve this problem, we have developed an efficient and green D-lactic acid fermentation process by using ammonia as neutralizer. The 106.7 g/l of D-lactic acid production and 0.89 g/g of consumed sugar were obtained by Sporolactobacillus inulinus CASD with a high optical purity of 99.7% by adding 100 mg/l betaine in the simple batch fermentation. The addition of betaine was experimentally proven to protect cells at high concentration of ammonium ion, increase D-lactate dehydrogenase specific activity and thus promote the production of D-lactic acid.

The aim of the study was the analysis of the occurrence of specific antibodies against HEV among hunters and foresters who are at risk to be exposed. The study group consisted of 210 hunters (23–80 years old) and 60 foresters (22–64 years old). Anti-HEV IgG were present in 3.81% of the samples of the hunters and in 5% of the samples of the foresters. The statistical analysis shows no significant differences in the results anti-HEV IgG between the groups of hunters and foresters (p = 0.5278). Significantly higher anti-HEV IgG titers were found in the older age group (> 55 years old).

The paper concerns the fungus Phoma complanata, isolated for the first time in Poland, from the roots and umbels of angelica (Archangelica officinalis) in 2009. The morphology of fungal isolates was tested on standard culture media. Moreover, the sequence analysis of ITS regions was conducted. Morphological similarity of P. complanata Polish isolates to the reference isolate obtained from CBS culture collection was determined and together with the molecular analysis confirmed the affiliation of the fungus to the species.