Inhibition of Zoonotic Pathogens Naturally Found in Pig Manure by Black Soldier Fly Larvae and Their Intestine Bacteria

Black soldier fly (BSF) larvae are often exposed to organic waste which harbors abundant zoonotic pathogens. We investigated the ability of BSF larvae to inhibit the zoonotic pathogens naturally found in pig manure. The zoonotic pathogens populations were detected by using selective medium during the conversion. Results showed that the viability of the zoonotic pathogens in pig manure was significantly affected. After eight days of conversion, the Coliform populations were undetected, and Staphylococcus aureus and Salmonella spp. decreased significantly on the eighth day. Antimicrobial assays of the purified recombinant defensin-like peptide 4 (DLP4) showed that this peptide exhibits inhibitory activity against S. aureus, Salmonella enterica serovar typhimurium, and Escherichia coli in vitro. Bacteria BSF-CL and BSF-F were isolated from the larvae gut, and both inhibited the growth of S. aureus and E. coli, but Salmonella spp. was sensitive to the BSF-CL strain (but not to the BSF-F strain). The results from our experiments indicate that BSF larvae are capable of functionally inhibiting potential zoonotic pathogens in pig manure through a variety of mechanisms including antimicrobial peptides expression and the gut associate microorganisms. This study provides a theoretical basis for further study on the combined mechanism of BSF larvae immunity and its gut microbes against the zoonotic pathogens in pig manure.

Isolation and Identification of Listeria Monocytogenes in Bangalore City from Various Food Samples

The current research emphasis on the isolation and differentiation of Listeria monocytogenes from different food samples most frequently infected with Listeriosis outbreaks. Crude chicken meat, raw milk, pasteurized cheese, ice cream and raw fish are samples from the city of Bangalore. The selective medium mainly used for the isolation of Listeria is oxford agar. Using isolated L. monocytogenes from food samples, morphologic and biochemical identification was carried out. 2 samples (fresh milk and Ice cream) were positive out of 5 samples; 3 samples (raw chicken meat, raw fish, and pasteurized cheese) were negative. The results conferred during this study indicate the contamination of Ice- cream and Raw Milk samples with L. monocytogenes.

OPTIMIZATION OF IN VITRO SELECTIVE MEDIA FOR SELECTING ANTHRACNOSIS-RESISTANT FLAX CELLS

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

On the assessment of the sources of inoculum of bacterial wilt in Brazil.

Dispersal of Ralstonia spp. cells by water and contaminated plant material and the importance of weeds as inoculum sources have been poorly investigated. Water of rivers, soil from fields of diverse crops and areas of natural vegetation both from the Amazonia, Cerrado and Mata Atlantica biomes, besides soil of the rhizosphere of weeds present in tomato fields with records of bacterial wilt were sampled and analyzed to detect Ralstonia spp. Seeds of tomato plants artificially and naturally infected with Ralstonia spp. were also processed. All samples were enriched a priori in selective medium South Africa (SMSA) and colonies were isolated in plates containing solid SMSA. Detection of Ralstonia spp. was confirmed by polymerase chain reaction with specific primers. The Co-operational PCR (CO-PCR) was also used to detect Ralstonia spp. Colonies were obtained from soil samples and from a commercial substrate sample. Five soil samples from eggplant fields, one from coffee field, one substrate from potato seed tuber production, two soil samples from the rhizosphere of Amaranthus spp., one from Bidens pilosa and one from Solanum americanum tested positive for Ralstonia spp. Besides these soil samples, five water samples of rivers were positive for CO-PCR detection: two samples from Amazonia, one from Cerrado and two samples from irrigation water collected from tomato fields located in the Mata Atlantica biome. Ralstonia spp. were not detected in tomato seeds. These results revealed potential inoculum sources, especially weeds, in areas with historical records of bacterial wilt. Additionally, rivers may act as dispersal agents of inoculum of Ralstonia spp.

Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda

Bovine brucellosis is endemic in Rwanda, although, there is paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n=300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigación y Tecnología Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The RBT seroprevalence was 20.7% (62/300), and 2.9% (8/300) with i-ELISA and 2.9% (8/300) using both tests in parallel. Brucella specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3) and B. melitensis (n=5) while Bruce-ladder PCR also identified B. abortus (n=5) and B. melitensis (n=6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures which is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen the national bovine brucellosis control program through vaccination as well as test- and-slaughter.

A Selective Medium for the Recovery and Enumeration of Fomitopsis meliae, Causing Lemon Canker and Wood Rot

A selective medium (FMS medium) was developed for the isolation and quantification of Fomitopsis meliae, the causal agent of lemon canker and brown wood rot, from plants, soil, and air. The optimal concentration and combination of fungicides and antibiotics was evaluated to determine the most selective condition for growing F. meliae. The resultant composition of the medium (FMS) per litre (pH 3.5) was: 16 mg thiophanate-methyl, 8 mg dichloran, 5 mg 2-phenylphenol, 100 mg fluopyram, 0.5 mg fludioxonil, 100 mg chloramphenicol, 100 mg streptomycin, 15 g malt extract, 2.5 g mycological peptone, and 15 g agar. The fungus was successfully isolated and enumerated from air, soil and plant tissues using FMS medium. Furthermore, FMS medium almost completely inhibited the growth of other plant pathogenic fungi, soil and air saprophytes. This selectivity was high enough to estimate spore inoculum of F. meliae in an air sample or as a spore trapping device in commercial lemon orchards. FMS medium will be useful for studying epidemiology and management of F. meliae.

Superior Non-Invasive Glucose Sensor Using Bimetallic CuNi Nanospecies Coated Mesoporous Carbon

The assessment of blood glucose levels is necessary for the diagnosis and management of diabetes. The accurate quantification of serum or plasma glucose relies on enzymatic and nonenzymatic methods utilizing electrochemical biosensors. Current research efforts are focused on enhancing the non-invasive detection of glucose in sweat with accuracy, high sensitivity, and stability. In this work, nanostructured mesoporous carbon coupled with glucose oxidase (GOx) increased the direct electron transfer to the electrode surface. A mixed alloy of CuNi nanoparticle-coated mesoporous carbon (CuNi-MC) was synthesized using a hydrothermal process followed by annealing at 700 °C under the flow of argon gas. The prepared catalyst’s crystal structure and morphology were explored using X-ray diffraction and high-resolution transmission electron microscopy. The electrocatalytic activity of the as-prepared catalyst was investigated using cyclic voltammetry (CV) and amperometry. The findings show an excellent response time of 4 s and linear range detection from 0.005 to 0.45 mM with a high electrode sensitivity of 11.7 ± 0.061 mA mM cm−2 in a selective medium.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6’)-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Antibiotic Resistance Profiling of Salmonella sp. isolated from African Catfish (Clarias gariepinus)

The African catfish (Clarias gariepinus) is an important fresh water fish consumed by a large percentage of the populace globally and it may be contaminated by pathogenic bacteria such as Salmonella sp. In this study, a total of fifteen (15) samples of African catfish were collected from different markets in Lokoja, Nigeria. The Salmonella sp. were isolated from the catfish samples by pre-enrichment in peptone water and subsequent inoculation on selective medium namely brilliant-green agar (BGA), bismuth sulphite agar (BSA) and Salmonella-Shigella agar (SSA). The Salmonella isolates were tested for susceptibility to 10 different commercially available antibiotics using the disc diffusion method. A total of thirty-four Salmonella species was isolated. The percentage occurrence of Salmonella sp. in the catfishes examined was very high (80%). The incidence of Salmonella sp. in the intestine (86.7%) of the catfish was higher than for the gills (66.7%) and the skin (73.3%). Majority of the isolates were resistant to Amoxicillin, Sulfomethoxazole-trimethoprim, Amoxicillin-clavulanic acid and Streptomycin. This study therefore demonstrated the occurrence of Salmonella species in African catfish with some exhibiting antibiotic resistance. Thus, there is a potential risk of transmission of drug resistant Salmonella species to man when contaminated catfish is consumed. The use of antibiotics in fish farming should be regulated so as to decrease antibiotic residues in fish.

The estimation of the viable count of mutans streptococcus in waterpipe smokers and cigarette smokers

Background: Waterpipe and cigarette are two products of tobacco consumption, tobacco use has detrimental effects on the oral cavity, numerous studies around the world have reported a significant relationship between smoking and increase dental caries and viable count of cariogenic bacteria, Materials and Methods: unstimulated saliva was collected from 84 subjects and divided equally into three groups waterpipe smokers, cigarette smokers, and non-smokers all of the participants are adult male aged between 25-60 years, dental caries was measured by use DMFT index, while S.mutans and S.sobrinus were isolated by using a selective medium SB 20M (Sugar bacitracin-20 modified) agar Results: this present study showed a significant (p≤0.01). higher DMFT, DT, MT, and FT among cigarette smokers group than both waterpipe smokers and non-smokers groups. The viable count of S mutans was significantly (p≤0.01).higher in the cigarette smokers group followed by the waterpipe smokers group and then the non-smoker's group, while the viable count of S sobrinus showed no statistical differences (P>0.05) between groups. The correlation of DMFT with S.mutans, and S sobrinus count, were significantly positive (p ≤0.05), in the cigarette smokers group only Conclusion: Dental caries increase in cigarette smokers, where the DMFT and S mutans viable count are less affected by waterpipe than cigarette smoking, a positive correlation is found between DMFT and .mutans streptococcus count only in cigarette smokers