Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A

Antimicrobial resistance in pathogenic bacteria is considered a major public health issue necessitating the discovery of alternative antimicrobial compounds. In this regard, targeted genome mining in bacteria occupying under-explored ecological niches has the potential to reveal such compounds, including bacteriocins. In this study, we determined the bacteriocin biosynthetic potential of the psychrophilic Clostridium estertheticum complex (CEC) through a combination of genome mining and phenotypic screening assays. The genome mining was performed in 40 CEC genomes using antiSMASH. The production of bacteriocin-like compounds was phenotypically validated through agar well (primary screening) and disk diffusion (secondary screening) assays using cell free supernatants (CFS) and partially purified extracts, respectively. Stability of four selected CFS against proteolytic enzymes, temperature and pH was determined while one CFS was analyzed by HRMS and MS/MS to identify potential bacteriocins. Twenty novel bacteriocin biosynthetic gene clusters (BBGC), which were classified into eight (six lantibiotics and two sactipeptides) distinct groups, were discovered in 18 genomes belonging to C. estertheticum (n = 12), C. tagluense (n = 3) and genomospecies2 (n = 3). Primary screening linked six BBGC with narrow antimicrobial activity against closely related clostridia species. All four preselected CFS retained activity after exposure to different proteolytic, temperature and pH conditions. Secondary screening linked BBGC1 and BBGC7 encoding a lantibiotic and sactipeptide, respectively, with activity against Bacillus cereus while lantibiotic-encoding BBGC2 and BBGC3 were linked with activity against B. cereus, Staphylococcus aureus (methicillin-resistant), Escherichia coli and Pseudomonas aeruginosa. MS/MS analysis revealed that C. estertheticum CF004 produces cesin A, a short natural variant of nisin, and HRMS indicated the production of a novel sactipeptide named estercticin A. Therefore, we have shown the CEC, in particular C. estertheticum, is a source of novel and stable bacteriocins that have activities against clinically relevant pathogens.

Validity and Reliability of the Persian Versions of Primary and Secondary Screening Instrument for Targeting Educational Risk Questionnaires

Background and Aim: Hearing loss in children leads to speech and language delays, low academic achievement, literacy delays, and psychosocial difficulties. Screening instrument for targeting educational risk (SIFTER) is one of the questionnaires used for evaluation of students’ performance in schools. The current study aims to develop Persian versions of primary and secondary SIFTER questionnaires and assessing their validity and reliability. Methods: The main English versions of primary and secondary SIFTER questionnaires were translated into Persian named as P-SIFTER and secondary P-SIFTER. Then, their face validities were determined based on the options of related experts. The final versions were completed by 55 teachers of 150 students (64 primary and 86 secondary school students) divided into two groups of hearing-impaired (HI) and normal-hearing (NH) students. The test- retest reliabilities were assessed in 117 students (64 primary and 53 secondary school students). Results: The results revealed that these questionnaires had high face validity. The content validity index for P-SIFTER and secondary P-SIFTER were obtained 0.94 and 0.92, respectively. The total score of P-SIFTER was 51.85 and 65.41 in HI and NH students, respectively. For the secondary P-SIFTER, it was 58.75 and 67.48, respectively. The test-retest reliability showed high correlation for NH and HI students between P-SIFTER and secondary P-SIFTER scores. The Cronbach’s alpha value for the overall score of P-SIFTER was 0.96 for both HI and NH students; for secondary P-SIFTER, the values were 0.94 and 0.93, respectively. Conclusion: The Persian versions of primary and secondary SIFTER questionnaires have acceptable validity and reliability.

A Comparison of Antibacterial Potential of Different Soil Actinomycetes from Pokhara

Actinomycetes are widely distributed in the environment and used for the production of several important secondary metabolites like antibiotics, immunosuppressive agents, enzymes and antitumor agents. Therefore, the main objective of this study is to isolate and assess antibacterial potential of different actinomycetes obtained from different soil samples. This study was conducted in the microbiology laboratories of Prithvi Narayan Campus and Lambda Food Lab Pvt Ltd, Pokhara. A total of nine soil samples were collected from different places of Pokhara (forest land, agriculture land and lake bank) and processed. Isolated actinomycetes were screened by primary and secondary screening for antibiotic producers against test organisms like Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Bacillus spp, E coli (ATCC 25922). This study isolated 27 actinomycetes in total, using the soil samples through spread plating on Starch Casein Agar (SCA) and by serial dilution. After incubation, actinomycetes colonies (rough, chalky) were selected for gram staining to observe thin thread-like mycelial and hyphal structures. The highest number of actinomycetes isolates were obtained from agricultural land’s soil samples (14 out of 27 isolates i.e. 51.85%) whereas only 3 isolates were obtained from the lake soil. Primary screening was performed on Nutrient agar where test bacteria were streaked perpendicular to the isolated actinomycetes to observe antagonism. This showed 12 actinomycetes as active isolates inhibiting at least one test bacteria. The antibacterial compounds were extracted by ethyl acetate method and used in secondary screening. Secondary screening in Mueller Hinton agar (MHA) further revealed five isolates showed promising inhibitory capacity. In both screening methods higher sensitivity was observed towards Gram-positive bacteria especially S aureus (ATCC 25923), and the least sensitivity towards Gram-negative bacteria especially Pseudomonas aeruginosa (ATCC 27853). Agricultural land was shown to harbor more actinomycetes than forest land and lake bank soil. Though variations were observed in primary and secondary screening, actinomycetes obtained from agricultural land demonstrated an inhibitory action against the Gram-positive and Gram-negative test organisms. As compared to Gram-negative bacteria, the Gram-positive had higher effects. These findings showed that soil of different locations of Pokhara valley found many actinomycetes strains, preventing the growth of pathogenic bacteria of certain kinds. The study suggested that further investigations need to be done that helped obtain new antimicrobial agents from actinomycetes, using various other sources.

Targeted Secondary Screening for Congenital Hypothyroidism in High-Risk Neonates: A 9 Year Review in a Large California Health Care System

Secondary screening for missed congenital hypothyroidism (CH) has been introduced sporadically, but its necessity and optimal strategy have not been recognized. We hypothesized that a simple clinical protocol (performed by a medical group without a governmental mandate) targeting infants at high risk for missed CH can identify cases. We performed a 9-year retrospective review of 338,478 neonates within a California health plan following the introduction of thyrotropin (TSH) secondary screening for neonates at high risk for missed CH due to very-low-birthweight (VLBW), hospitalized congenital heart disease (CHD), and same-sex multiples (SSM). Screening performance by day 60 of life was 95% successful for VLBW and >50% for CHD and SSM, leading to an additional 35% CH treated cases despite re-testing only 1.7% of the cohort. Infants with VLBW or CHD were 33 times more likely (190 times more likely for CHD with Down Syndrome) to receive treatment for CH than random infants diagnosed by primary screening (p < 0.001), and 92% of these infants were not found by primary newborn screening. Currently, permanent disease has been documented in 84% of CH by primary screening compared to 27% by secondary screening (p < 0.001). This targeted secondary screening program identifies and treats additional CH cases after TSH-only newborn screening.

Antibacterial Activity of Lactic Acid Bacteria Isolated from Fermented Ethiopian Traditional Dairy Products against Food Spoilage and Pathogenic Bacterial Strains

Fermented Ethiopian traditional dairy products containing LAB that show antibacterial activities against various food spoilage and pathogenic bacteria have been used for the preservation of fermented dairy products for a long time. However, there are no comprehensive scientific reports on the antibacterial activity of LAB isolated from various fermented dairy products in Pawe Woreda. The objective of the study was to evaluate the antibacterial activity of LAB isolated from traditionally fermented Ethiopian dairy products against spoilage and pathogenic bacteria. Thirty-five samples of fermented dairy products were collected from three cattle-farming areas of Pawe Woreda. A total of 97 LAB were isolated and screened primarily using the perpendicular streak plate method against 3 Gram-positive and 3 Gram-negative bacterial strains. Out of the 97 strains, 10 were active against at least two of the tested bacteria, of which 7 strains were selected for secondary screening by their broad-spectrum antibacterial activities. The seven in vitro antibacterial activities of the extract ranged from 5 to 16 mm in diameter during the secondary screening. In this study, Z2, Z4, and N2 strains exhibited the highest inhibition zone with broad-spectrum activity against all tested bacteria. The MIC and MBC values range from 0.10 to 0.30 µg/µL and 0.20 to 0.50 µg/µL, respectively. Following morphological, physiological, biochemical, and molecular characteristics, seven potent strains were identified as Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Leuconostoc mesenteroides, Streptococcus thermophilus, and Lactococcus lactis. According to the findings of this study, Ethiopian fermented dairy products were the most potent source of bioactive compounds with potential effects against food spoilage and pathogenic bacterial strains.

The screening of heart disease by cardiac auscultation and electrocardiography examination in pregnant women in Puskesmas Tegalrejo, Yogyakarta, Indonesia

Women adapt to pregnancy through multi-organ system physiologic changes, including cardiovascular adaptations. These changes affect those with pre-existing cardiovascular problems differently, and subsequently lead to higher probability of death caused by cardiovascular diseases during pregnancy. Therefore, detection of cardiovascular disease early in pregnancy is important to lower maternal morbidity and mortality by providing prompt and adequate management. This study aimed to evaluate and test the feasibility of integrating 12-lead electrocardiogram (ECG) examination and antenatal care (ANC) screening as a simple and effective method for early detection of heart abnormality in pregnant woman. Pregnant women were recruited in this study in any trimester who attended ANC for a routine pregnancy examination in Puskesmas Tegalrejo Yogyakarta. The subjects underwent primary screening which focused on cardiac auscultation and 12-lead ECG examinations, and those who had abnormal findings were further followed-up in secondary screening by using trans-thoracic echocardiography to confirm heart abnormality. A total of 523 pregnant women from Puskesmas Tegalrejo were included in this study. 15 (2.8%) pregnant woman were suspected to have heart abnormalities; from those, 3 (0.5%) were found with heart murmurs with abnormal ECG readings, 1 (0.19%) had heart murmurs with normal ECG results, and 11 (2.1%) had abnormal ECG readings only. The secondary screening of those patients resulted in 1 (0.19%) pregnant woman who was diagnosed with Atrial Septal Defect. Our study found that among 15 patients identified with suspected ECG abnormalities, one mother who underwent ANC was newly diagnosed with a pre-existing cardiac abnormality. Our study concluded this screening method is a simple and feasible integrated heart screening program that can be implemented widely. We hope this integrated heart screening program may benefit pregnant women who may have cardiac abnormalities to be detected as early as possible, thus reducing maternal morbidity and mortality.

Primary, secondary screening and evaluation of LAS degrading potential of soil bacteria isolated from Vidya Pratishthan’s college area

Inefficiency of use of soap in hard water popularized the use of anionic surfactants namely, branched alkylbenzene sulphonates (BAS) and Linear alkyl benzene sulphonates (LAS) in detergents. The low cost and superior solubility in hard water had made them popular and first choice of many household care-products. However, the disadvantage associated with their use was formation of stable foam over waterbodies which is detrimental for both the aquatic plants and animals. The current study involves primary and secondary screening of LAS degrading organisms and evaluates their potential. The three isolates screened isolate 1 can degrade LAS to the tune of 66% in 5 days, isolate 2 and isolate 3 degrade 72.16% and 68.25% respectively in 4 days.

Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent

Background A wide variety of bacterial species produces protease enzyme, and the application of the same enzyme has been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen, and identify alkaline protease-producing bacteria that were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia. Purpose To isolate and characterize the alkaline protease-producing bacteria from leather industrial effluents. Methods Samples are collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolated protease-producing bacteria using skim milk agar media. After studying primary and secondary screening using zonal inhibition methods to select potential protease-producing bacteria using skim milk agar media. Finally, to identify the potential bacteria using biochemical methods, bacterial biomass, protease activity, and gene sequencing (16S rRNA) method to finalize the best alkaline protease producing bacteria identified. Results First twenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at the Modjo town of Ethiopia. The isolated bacteria were screened using the primary and secondary screening method with skim milk agar medium. At the primary level, we selected three isolates namely ML5(14 mm), ML12(18 mm), and MS12 (15 mm), showing the highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to primary screening. Further secondary screening confirmed that the zone of inhibition methods ML5 (14.00±0.75 mm), ML12 (19.50±0.66 mm), and MS12 (15.00±1.32 mm) has efficient proteolytic activity and can be considered as effective protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species, and all the three bacterial isolates were found out to be of Bacillus species. The shake flask method was carried out to identify the most potent one having greater biomass production capabilities and protease activity. ML12 isolated from leather effluent waste showed the highest protease activity (19 U/ml), high biomass production, and the same was subjected to molecular identification using 16s sequencing and a phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 (Bacillus cereus strain -MN629232.1) is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1). Conclusions This study has exposed that from twenty-eight different bacterial samples isolated from leather industry effluent; further primary and secondary screening methods were selected three potential alkaline protease strains. Finally, based on its biochemical identification, biomass, and protease activity, ML12 (Bacillus cereus strains) is the best strain identified. The alkaline protease has the significant feature of housing potent bacterial species for producing protease of commercial value.

Plasma Globotriaosylsphingosine and α-Galactosidase A Activity as a Combined Screening Biomarker for Fabry Disease in a Large Japanese Cohort

Fabry disease is an X-linked disorder of α-galactosidase A (GLA) deficiency. Our previous interim analysis (1 July 2014 to 31 December 2015) revealed plasma globotriaosylsphingosine as a promising primary screening biomarker for Fabry disease probands. Herein, we report the final results, including patients enrolled from 1 January to 31 December 2016 for evaluating the potential of plasma globotriaosylsphingosine and GLA activity as a combined screening marker. We screened 5691 patients (3439 males) referred from 237 Japanese specialty clinics based on clinical findings suggestive of Fabry disease using plasma globotriaosylsphingosine and GLA activity as primary screening markers, and GLA variant status as a secondary screening marker. Of the 14 males who tested positive in the globotriaosylsphingosine screen (≥2.0 ng/mL), 11 with low GLA activity (<4.0 nmol/h/mL) displayed GLA variants (four classic, seven late-onset) and one with normal GLA activity and no pathogenic variant displayed lamellar bodies in affected organs, indicating late-onset biopsy-proven Fabry disease. Of the 19 females who tested positive in the globotriaosylsphingosine screen, eight with low GLA activity displayed GLA variants (six classic, two late-onset) and five with normal GLA activity displayed a GLA variant (one classic) and no pathogenic variant (four late-onset biopsy-proven). The combination of plasma globotriaosylsphingosine and GLA activity can be a primary screening biomarker for classic, late-onset, and late-onset biopsy-proven Fabry disease probands.