Staphylococcus aureus Bacteriophage 52 Endolysin Exhibits Anti-Biofilm and Broad Antibacterial Activity Against Gram-Positive Bacteria.

Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.

Genome-based classification of Streptomyces anatolicus sp. nov., an actinobacterium with antimicrobial and cytotoxic activities, and reclassification of Streptomyces nashvillensis as a later heterotypic synonym of Streptomyces tanashiensis.

During the course of isolating novel actinobacteria producing bioactive metabolites, strain BG9HT was obtained from an arid soil sample in Erzurum, Turkey. Pairwise sequence comparisons for 16S rRNA gene sequences showed the strain was a member of the genus Streptomyces and it shared the highest 16S rRNA gene sequence identity of 99.7% with Streptomyces huasconensis HST28T. Comparative genome analyses based on digital DNA-DNA hybridization and average nucleotide identity revealed that strain BG9HT represents a novel species within the genus Streptomyces. The polyphasic analysis also confirmed that the strain has typical characteristics of the genus Streptomyces. The strain has LL-diaminopimelic acid as diagnostic amino acid, and galactose, mannose and trace amounts of glucose and ribose as whole-cell sugars. Polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, unidentified aminolipids, phospholipids and lipids. Major isoprenoid quinones were MK-9(H6), MK-9(H4), and MK-9(H8). Its genome size is approximately 7.2 Mb with 71.2% G+C content. The methanolic extract of strain BG9HT showed antimicrobial and cytotoxic activities. Further genomic analyses of strain BG9HT confirmed its high potential to produce novel secondary metabolites. On the basis of phenotypic and phylogenetic analyses, strain BG9HT represents a novel species of the genus Streptomyces, for which Streptomyces anatolicus sp. nov. is proposed, and it holds high promise for novel biosynthetic metabolites of value to the biopharmaceutical industry. We also propose Streptomyces nashvillensis as a later heterotypic synonym of Streptomyces tanashiensis as a result obtained through analysis of overall genome relatedness indices.

A comparative study of antimicrobial, anti-quorum sensing, anti-biofilm, anti-swarming, and antioxidant activities in flower extracts of pecan (Carya illinoinensis) and chestnut (Castanea sativa).

Antibiotic resistance, which has increased rapidly in recent years because of uncontrolled and unconscious antibiotic consumption, poses a major threat to public health. The inadequacy of existing antibiotics has increased the need for new, effective, and less toxic antibiotic raw materials or antibiotic derivatives. Pecan (Carya illinoinensis) and Chestnut (Castanea sativa) flowers possess abundant pollen contents and exhibit similar morphological features. The purpose of this study was to compare these two flower extracts in terms of their antimicrobial and antioxidant activities. Total phenolic content, total flavonoid contents, and phenolic components were also analyzed in aquatic and ethanolic extracts. Antioxidant activities were measured using ferric reducing/antioxidant capacity (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Antimicrobial and antifungal activities were compared by means of agar diffusion tests against bacteria including Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis, Acinetobacter haemolyticus, and Chromobacterium violaceum, and the yeasts Candida albicans and Candida parapsilosis. Anti-quorum sensing (anti-QS), anti-biofilm, and anti-swarming (SW) activities were also studied against Chromobacterium violaceum ATCC 31532, Chromobacterium violaceum ATCC 12472, and Pseudomonas aeruginosa PA01, respectively. Both extracts were rich in ellagic acid and gallic acid and exhibited similar antioxidant properties. Both flower extracts exhibited high antimicrobial and antifungal activities as well as anti-biofilm, anti-QS, and anti-SW activities.

Prebiotic potential of enzymatically prepared resistant starch in reshaping gut microbiota and their respond to body physiology.

The increase in consumer demand for high-quality food products has led to growth in the use of new technologies and ingredients. Resistant starch (RS) is a recently recognised source of fibre and has received much attention for its potential health benefits and functional properties. However, knowledge about the fate of RS in modulating complex intestinal communities, the microbial members involved in its degradation, enhancement of microbial metabolites, and its functional role in body physiology is still limited. For this purpose, the current study was designed to ratify the physiological and functional health benefits of enzymatically prepared resistant starch (EM-RSIII) from maize flour. To approve the beneficial health effects as prebiotic, EM-RSIII was supplemented in rat diets. After 21 days of the experiment, EM-RSIII fed rats showed a significant reduction in body weight gain, fecal pH, glycemic response, serum lipid profile, insulin level and reshaping gut microbiota, and enhancing short-chain fatty acid compared to control. The count of butyrate-producing and starch utilizing bacteria, such as Lactobacillus, Enterococcus, and Pediococcus genus in rat's gut, elevated after the consumption of medium and high doses of EM-RSIII, while the E. coli completely suppressed in high EM-RSIII fed rats. Short-chain fatty acids precisely increased in feces of EM-RSIII feed rats. Correlation analysis demonstrated that the effect of butyrate on functional and physiological alteration on the body had been investigated during the current study. Conclusively, the present study demonstrated the unprecedented effect of utilising EM-RSIII as a diet on body physiology and redesigning gut microorganisms.

Immune responses in multiple hosts to Nucleocapsid Protein (NP) of Crimean-Congo Hemorrhagic Fever Virus (CCHFV).

In 2019, the World Health Organization declared 3 billion to be at risk of developing Crimean Congo Hemorrhagic Fever (CCHF). The causative agent of this deadly infection is CCHFV. The data related to the biology and immunology of CCHFV are rather scarce. Due to its indispensable roles in the viral life cycle, NP becomes a logical target for detailed viral immunology studies. In this study, humoral immunity to NP was investigated in CCHF survivors, as well as in immunized mice and rabbits. Abundant antibody response against NP was demonstrated both during natural infection in humans and following experimental immunizations in mice and rabbits. Also, cellular immune responses to recombinant NP (rNP) was detected in multispecies. This study represents the most comprehensive investigation on NP as an inducer of both humoral and cellular immunity in multiple hosts and proves that rNP is an excellent candidate warranting further immunological studies specifically on vaccine investigations.

[Comparison of the modified Hodge test and the Carba NP test for detection of carbapenemases in Enterobacteriaceae isolates]. / Enterobacteriaceae izolatlarinda karbapenemazlarin saptanmasinda modifiye Hodge testi ve Carba NP testlerinin karsilastirilmasi.

A rapid, practical, and accurate identification of carbapenemase-producing Enterobacteriaceae isolates is crucial for the implementation of appropriate infection control measures and proper treatment of the infections. For this purpose, a large number of phenotypic test methods have been developed, although none has 100% sensitivity and specificity. Variations in sensitivity and specificity of these tests based on the type of beta-lactamase enzymes carried by that isolates might result in differences between regions and countries. The aim of this study was to compare the performances of widely used modified Hodge test (MHT) and Carbapenemase Nordmann-Poirel (Carba NP) test in the detection of carbapenemases in Enterobacteriaceae family members. A total of 65 Enterobacteriaceae isolates (43 bla(OXA-48), 10 bla(VIM), 9 bla(IMP), 1 bla(NDM-1), 1 bla(KPC-2) and 1 bla(OXA-48)+bla(VIM) carrying strains) that showed decreased sensitivity to at least one carbapenem (ertapenem, imipenem or meropenem), and carriage of carbapenemase gene confirmed by polymerase chain reaction (PCR), were included in the study. Seventy-eight isolates showing decreased susceptibility to carbapenems but lacking carbapenemase genes were used as controls. All isolates were identified by using conventional methods as well as automated BD Phoenix System (Becton Dickinson, USA). The antimicrobial susceptibility testing was performed using the same automated system, and was confirmed by disk diffusion method. Results were evaluated according to the CLSI criteria. MHT was performed in accordance with the CLSI guideline, and Carba NP test was carried out by a modified protocol. Instead of imipenem monohydrate, which was used in the original protocol, 6 mg/ml imipenem/cilastatin was used in the modified protocol. In the study, MHT identified 90.8% (59/65) of carbapenemase-producing isolates, while 93.9% (61/65) of the isolates were identified by Carba NP test. With MHT, four Klebsiella pneumoniae producing OXA-48, one Escherichia coli producing IMP, and one K.pneumoniae producing NDM-1, and with Carba NP test, one E.coli and one K.pneumoniae producing OXA-48, one E.coli producing IMP, and one Enterobacter cloacae producing VIM could not de detected. Three OXA-48-producing isolates (two K.pneumoniae and one E.coli) yielded late and weak positive results with Carba NP test. MHT had false positivity for 31 isolates, while Carba NP test showed no false positivity. In comparison of the sensitivity and specificity of the two tests, sensitivities were found to be similar although the Carba NP has a slightly higher sensitivity than the MHT (93.9% versus 90.8%, respectively; p= 0.754), Carba NP was found more specific (100% versus 60.3%, respectively; p< 0.0001). With Carba NP test, 26% of the isolates (n= 16) were positive within 15 minutes, and 85% (n= 52) were positive within the first hour. It was concluded that, Carba NP test showed high sensitivity and specificity than the MHT and the results can be obtained more rapidly for the presence of carbapenemases in Enterobacteriaceae. The use of MHT alone is not recommended to confirm the presence of carbapenemases produced by Enterobacteriaceae. On the other hand Carba NP test can be used for this purpose, however molecular analysis should be considered for suspicious negative results.

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae / Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

The extended-spectrum â-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15 percent) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital

O isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL) está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram identificadas pelo teste de sinergia em disco-duplo (DDS). Foram detectadas vinte cepas produtoras de ESBL, entre as quais Escherichia coli (n=9), Klebsiella pneumoniae (n=7), Klebsiella oxytoca (n=2) e Enterobacter aerogenes (n=2), que foram posteriormente analisadas quanto a suas características de transferência de resistência, perfil plasmidial e natureza dos genes de resistência. Os testes de transferência de plasmídios foram realizados empregando técnicas de conjugação em caldo. Os genes TEM e SHV foram analisados pela reação da polimerase em cadeia (PCR) e hibridização com sondas especificas. A detecção de plasmídios epidêmicos foi feita por análise dos plasmídios R com a enzima de restrição EcoRI. Através desta análise, foram obtidos catorze perfis plasmidiais (A, B1, B2, C1 e C2 até L).Observou-se pela PCR que a maioria dos plasmidios carregavam genes derivados de TEM e SHV, confirmados através da detecção dos genes pelos testes de hibridização. As evidencias epidemiológicas indicaram que havia uma aparente transferência horizontal dos plasmídios R conjugativos entre as enterobactérias multiresistentes neste hospital.

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae.

The extended-spectrum ß-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15%) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital.

[Biotypes and antibiotic resistance patterns of Gardnerella vaginalis strains isolated from healthy women and women with bacterial vaginosis]. / Saglikli ve bakteriyel vajinozlu kadinlardan izole edilen Gardnerella vaginalis suslarinin biyotiplendirilmesi ve antibiyotik direnç durumlarinin belirlenmesi.

As Gardnerella vaginalis is accepted as a member of normal vaginal flora, it is one of the dominant species which has been related to bacterial vaginosis (BV). The aim of this study was to determine the isolation rate, biotypes and antibiotic resistance patterns of G.vaginalis from the vaginal swab samples of 408 women who were admitted to the outpatient clinics of Family Planning Center. Hippurate hydrolysis, lipase and beta-galactosidase tests were performed for biotyping the isolates, and agar dilution (for metronidazole) and disk diffusion (for clindamycin) tests were used for the detection of antibiotic resistance patterns. As a result, by Nugent's BV scoring protocol, 122 (29.9%), 20 (29.4%), 137 (33.6%), and 18 (4.4%) of the women were diagnosed as BV, intermediate form, normal vaginal flora (NVF) and mycotic vaginosis, respectively. The overall isolation rate of G.vaginalis was found as 23% (94/408). Of them, 56.4% (53/94) and 8.5% (8/94) were isolated from samples of BV cases and subjects with NVF, respectively, and the difference was statistically significant (p<0.05). The biotyping results showed that the most frequently detected types were biotype 1 (44%), 5 (20%) and 4 (18%). There was no statistically significant difference between the biotype distribution of BV patients and the subjects who have NVF (p=0.687). The results of antibiotic susceptibility tests indicated that 70% and 53% of the isolates were resistant to metronidazole and clindamycin, respectively. It was of interest that MIC values for metronidazole was > or =128 microg/ml in 57% of resistant strains. The data of this study has emphasized that the metronidazole resistance is very high in our population, and the large scale studies are needed to clarify the relationship between BV and G.vaginalis biotypes, which can be found in the normal vaginal flora.

[Comparison of vaginal lactobacilli and Lactobacillus species discrimination using classic methods and polymerase chain reaction]. / Vajinal laktobasiller ve laktobasillerin tür ayiriminda klasik yöntemler ile polimeraz zincir reaksiyonunun karsilastirilmasi.

The aim of this study was to identify the vaginal lactobacilli in the species level and to investigate the concordance between classical methods and polymerase chain reaction (PCR) for the typing of these isolates. Vaginal swab samples which have been collected from women who were admitted to the outpatient clinics of Gynecology Department of our hospital, were examined by standard microbiological methods and additionally were inoculated into selective lactobacilli media. Of 200 subjects, 59.5% have had normal vaginal flora, 31% were diagnosed as bacterial vaginosis and 9.5% as vaginal candidiasis. The lactobacilli isolation rates of these groups were found 76.5%, 45.2% and 78.9%, respectively. A total of 160 facultative anaerobic Lactobacillus strains were isolated from 134 (67%) of the swab samples. Of these, 90.6% were identified into species level by classical methods, and the most frequently isolated species in our study was found as L. gasseri (40%), followed by L. delbrueckii (18%). The comparative study was performed only for 66 isolates, and 58 of them (87.8%) were grouped into 7 species while 8 have not been identified by classical methods, however, all of 66 isolates were successfully grouped into 4 species by PCR. Fourty-five of 66 strains have been found to be identical by means of classical methods and PCR, and the concordance between the methods were found 68.2 percent.