Folia Microbiologica

Infection caused by Klebsiella pneumoniae ST11 in a patient after craniectomy Abstract Klebsiella pneumoniae infections have always been an important problem in public health, but today, the increasing resistance of these bacteria to antibiotics due to β-lactamases production has renewed interest in K. pneumoniae infections. The aim of the study was to present a case of a neurosurgical patient with multidrug-resistant K. pneumoniae ST11 infection after craniectomy. Four K. pneumoniae isolates from various clinical materials of the patient undergone identification and susceptibility testing with the Vitek2 system. Tests for β-lactamases production were performed according to EUCAST guidelines. Strains were analyzed for bla genes responsible for β-lactamase production (blaTEM, blaSHV, blaCTX-M, blaVIM, blaIMP, blaNDM, blaKPC, blaOXA-48) using PCR. Moreover, the genetic relatedness of these isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All tested strain presented multidrug resistance. The highest susceptibility was observed for imipenem, meropenem, and ertapenem. The strain isolated from the nervous system was ESBL-positive with blaSHV-11, blaTEM-1, and blaCTX-M-15 genes. Additionally, the strain from urine was blaKPC-3-positive. Molecular typing revealed that all strains belonged to the same clone and identified two PFGE profiles. The analysis of MLST allelic profile showed that tested K. pneumoniae strains belonged to ST11. Identification of ST11 K. pneumoniae as etiological factor of infection unfavorably impacts on prognosis among neurosurgical patient after craniectomy.

Effect of silver nanoparticles conjugated to thiosemicarbazide on biofilm formation and expression of intercellular adhesion molecule genes, icaAD , in Staphylococcus aureus Abstract Biofilm formation is regarded as an important factor in the establishment of infections caused by Staphylococcus aureus. In the present study, phenotypic and molecular assays were used to evaluate antibiofilm potential of thiosemicarbazide (Tsc) conjugated with silver nanoparticles (Ag NPs) and functionalized by glutamic acid (Ag@Glu/Tsc NPs) against methicillin-resistant S. aureus (MRSA). Ag NPs were synthesized using precipitation method and conjugated to Tsc using glutamic acid. The NPs were characterized using SEM and FTIR spectroscopy analyses. Then, antibiofilm potential of the prepared NPs against MRSA strains was evaluated using phenotypic method and their effects on the expression of biofilm-associated genes icaA and icaD. Finally, the genes involved with the synthesis of intercellular adhesion molecules were determined. According to the results, Ag@Glu/Tsc NPs inhibited biofilm formation of MRSA strains up to 76.7% compared with the control. In addition, expression of the biofilm-associated genes icaA and icaD reduced by 66.7% and 60.3%, respectively in the presence of sub-inhibitory concentration of Ag@Glu/Tsc NPs. In conclusion, Ag@Glu/Tsc NPs could be considered as a potent antibacterial agent to inhibit bacterial biofilms.

Comparative evaluation of structure and characteristic of peptidyl-prolyl cis-trans isomerase proteins and their function in Salmonella Typhimurium stress responses and virulence Abstract Peptidyl-prolyl cis-trans isomerases (PPIase) exhibit chaperone activity and assist in protein folding by increasing the rate of cis-trans transition on proline-peptide bonds. The current study aimed to identify and characterize three genes, ppiA, ppiB, and ppiC, which encode proteins of the PPIase family in the bacterium Salmonella enterica serovar Typhimurium. Salmonella Typhimurium is a facultative intracellular zoonotic pathogen that causes food- and water-borne gastroenteritis in humans (leading to bacteremia in immune-compromised subjects). Recombinant clones for the three genes were constructed and sequenced and the sequences submitted to NCBI GenBank. Three-dimensional structures for the corresponding proteins were predicted by comparative modeling. A maximum-likelihood phylogenetic gene tree constructed for the three genes showed a low evolutionary mean diversity, indicating strong evolutionary conservation. Further, single-gene deletion mutant strains, generated for the respective genes, were observed to be more susceptible to the stationary phase of growth and heat stress conditions and showed reduced survival within macrophage cells line. The present study thus indicates that ppiA, ppiB, and ppiC genes are conserved among Salmonella genome, are critical for the growth of Salmonella Typhimurium in the examined stress conditions, and may play a role in its responses and virulence.

Analysis of inflammatory cytokine expression in the urinary tract of BALB/c mice infected with Proteus ( P .) mirabilis and enteroaggregative Escherichia ( E .) coli (EAEC) strains Abstract This study aimed to analyze the proinflammatory cytokine mRNA expression in the urinary tract of BALB/c mice infected with bacterial strains with uropathogenic potential. Groups of four 6-week-old female BALB/c mice were intraurethrally inoculated with 5 × 107 colony-forming units (CFU) of P. mirabilis ATCC29906, EAEC O42, P. mirabilis RTX339, or sterile saline (control group) and then sacrificed at 0, 2, 4, 7, or 10 days post-infection (p.i.). Samples were cultured to determine the CFU/mL in urine or CFU/g in the bladders and kidneys. Cytokine expression (tumor necrosis factor (TNF)-α and interleukin (IL)-1β, -6, and -8) was evaluated in the target organs using real-time PCR and immunohistochemistry; histology was examined with hematoxylin and eosin staining. The results are presented as the means and standard deviations and were compared using one-way ANOVA, with p < 0.05 indicating significant differences. Bacteriuria was not detected in the infected groups; bacterial colonization occurred in the target organs at all time points, but was higher in mice infected with EAEC O42 or P. mirabilis RTX339 at 7 days p.i. The expression of all cytokine mRNAs was seen, but only the levels of the IL-8 protein increased in situ at 7 days p.i. in the P. mirabilis RTX339 and EAEC O42 groups in both organs. Morphological alterations, observed in all of the infected groups, were more prominent in the EAEC O42 and P. mirabilis RTX339 groups. The findings provide insights into the uropathogenicity and inflammatory cytokine expression in the urinary tract of mice infected with three previously untested bacterial strains.

Cellulolytic thermophilic microorganisms in white biotechnology: a review Abstract Enzymes of microbial origin are of immense importance for organic material decomposition leading to bioremediation of organic waste, bioenergy generation, large-scale industrial bioprocesses, etc. The market demand for microbial cellulase enzyme is growing more rapidly which ultimately becomes the driving force towards research on this biocatalyst, widely used in various industrial activities. The use of novel cellulase genes obtained from various thermophiles through metagenomics and genetic engineering as well as following metabolic engineering pathways would be able to enhance the production of thermophilic cellulase at industrial scale. The present review is mainly focused on thermophilic cellulolytic bacteria, discoveries on cellulase gene, genetically modified cellulase, metabolic engineering, and their various industrial applications. A lot of lacunae are yet to overcome for thermophiles such as metagenome analysis, metabolic pathway modification study, search of heterologous hosts in gene expression system, and improved recombinant strain for better cellulase yield as well as value-added product formation.

Phospholipid biosynthesis disruption renders the yeast cells sensitive to antifungals Abstract To understand the role of phospholipids on Cdr1p (drug exporter)-mediated drug resistance in yeast, the phospholipids biosynthesis genes PSD1, PSD2, CHO2, and OPI3 were deleted in a strain of Saccharomyces cerevisiae already overexpressing Cdr1-GFP of Candida albicans as a heterologous system. The effect of phospholipids biosynthesis gene deletion was analyzed on Cdr1p-GFP-mediated drug resistance as well as its localization. The results indicate that phospholipids biosynthesis disruption makes the cell sensitive to several drugs including fluconazole (FLC), with Δpsd1/Cdr1-GFP being worst affected. Interestingly, unlike sterols and sphingolipids, the localization of Cdr1p was unaffected by phospholipid biosynthesis gene disruption. Concomitantly, phospholipids mutants also showed an increase in reactive oxygen species (ROS) generation, as verified by fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method. In addition, the sensitivity of phospholipid mutants with FLC was found to be synergistic to ROS generation, resulting in further reduction of growth. Thus, this study proposes phospholipid biosynthesis as a novel target for antifungal therapy.

BAL fluid analysis in the identification of infectious agents in patients with hematological malignancies and pulmonary infiltrates Abstract The present study aims to evaluate the diagnostic yield of bronchoalveolar lavage (BAL) fluid in patients with hematological malignancies and describe the most common pathogens detected in BAL fluid (BALF.) An analysis of 480 BALF samples was performed in patients with hematological malignancies over a period of 7 years. The results of culture methods, PCR, and immunoenzymatic sandwich microplate assays for Aspergillusgalactomannan (GM) in BALF were analyzed. Further, the diagnostic thresholds for Aspergillus GM and Pneumocystis jiroveci were also calculated. Microbiological findings were present in 87% of BALF samples. Possible infectious pathogens were detected in 55% of cases; 32% were classified as colonizing. No significant difference in diagnostic yield or pathogen spectrum was found between non-neutropenic and neutropenic patients. There was one significant difference in BALF findings among intensive care units (ICU) versus non-ICU patients for Aspergillus spp. (22% versus 9%, p = 0.03). The most common pathogens were Aspergillus spp. (n = 86, 33% of BAL with causative pathogens) and Streptococcus pneumoniae (n = 46, 18%); polymicrobial etiology was documented in 20% of cases. A quantitative PCR value of > 1860 cp/mL for Pneumocystis jirovecii was set as a diagnostic threshold for pneumocystis pneumonia. The absorbance index of GM in BALF of 0.5 was set as a diagnostic threshold for aspergillosis. The examination of BAL fluid revealed the presence of pathogen in more than 50% of cases and is, therefore, highly useful in this regard when concerning pulmonary infiltrates.

Glutamine synthetase type I ( gln AI) represents a rewarding molecular marker in the classification of bifidobacteria and related genera Abstract The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.

Development of a sequence-characterized amplified region marker for detection of Ascochyta rabiei causing Ascochyta blight in chickpea Abstract Ascochyta blight of chickpea is caused by Ascochyta rabiei (Pass.) Labr. which is primarily seedborne. For rapid detection and precise identification of A. rabiei, a sequence-characterized amplified region (SCAR) marker was developed for detection of genomic DNA and infected plant DNA. An SSR primer amplified monomorphic band was cloned in pGEM®-T easy vector and sequenced. The best primer pair was selected and validated on A. rabiei. The specificity and sensitivity of the SCAR-based marker designated as MBAR was evaluated using conventional PCR and real-time PCR. The marker produced consistently an amplicon size of 196 bp in all A. rabiei isolates tested. The sensitivity of the marker was 0.1 ng of genomic fungal DNA and 0.5 ng of plant DNA by conventional PCR and 0.5 pg of A. rabiei DNA and 1.0 pg of plant DNA by real-time PCR. This is the first SCAR marker having high specificity and sensitivity towards A. rabiei. The marker may be useful in detecting the pathogen before the disease appearance and in plant quarantine program to detect the pathogen in seed lots.

Recommendations for cerebrospinal fluid analysis Abstract Diseases of the central nervous system (CNS) mean for the human organism a potentially dangerous situation. An investigation of cerebrospinal fluid (CSF) provides important information about a character of CNS impairment in the decision-making diagnostic and therapeutic algorithm. The authors present a brief overview of available cerebrospinal fluid assays, shortened indication criteria, a recommended algorithm of CSF assessment in different suspected diseases, and a view of the external quality system. The whole portfolio of obtainable CSF methodology is further subdivided according to the adequate choice into the first and inevitable basic routine panel, and following complicated analyses of highly specialized character. The basic panel is considered for standard laboratories, the complete specialized assessment should be provided by a super-consulting laboratory.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com