Search results for: pathogens

Authors: Memoona Ramzan, Bushra Tabassum, Anwar Khan, Muhammad Tariq, Mudassar Fareed Awan, Idrees Ahmad Nasir, Zahida Qamar, Naila Shahid, Tayyab Husnain

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Biological control is a novel approach being used in crop protection nowadays. Bacteria like Bacillus and Pseudomonas are reported for this purpose and few of their products are commercially available too. Rhizosphere and phyllosphere of healthy cotton plants were used as a source to isolate bacteria capable of exhibiting properties worthy for selection as biocontrol agent. For this purpose all isolated strains were screened for the activities like phosphate solubilization, Indole acetic acid (IAA) production and biocontrol against fungi. Two strains S1HL3 and S1HL4 showed phosphate solubilization and IAA production simultaneously while two other JS2HR4 and JS3HR2 were good inhibitors of fungal pathogens. Through biochemical and molecular characterization these bacteria were identified as P. aeruginosa, Burkholderia and Bacillus respectively. In green house trials of these isolates against Cotton leaf curl virus (CLCuV), seven treatments including individual bacterial isolate and consortia were included. Treated plants were healthy as compared to control plants in which upto 74% CLCuV symptomatic plants exist. Maximum inhibition of CLCuV was observed in T7 treated plants where viral load was only 0.4% as compared to control where viral load was upto 74%. This treatment consortium included Bacillus and Pseudomonas isolates; S1HL3, S1HL4, JS2HR4 and JS3HR2. Principal Component Biplot depicted highly significant correlation between percentage viral load and the disease incidence.

Keywords: cotton leaf curl virus, biological control, bacillus, pseudomonas

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Authors: Katarína Čurová, Leonard Siegfried, Radka Vargová, Marta Kmeťová, Vladimír Hrabovský

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Introduction: Cytolethal distending toxins (CDTs) represent intracellular acting proteins which interfere with cell cycle of eukaryotic cells. They are produced by Gram-negative bacteria with afinity to mucocutaneous surfaces and could play a role in the pathogenesis of various diseases. CDTs induce DNA damage probably through DNAse activity, which causes cell cycle arrest and leads to further changes (cell distension and death, apoptosis) depending on the cell type. Five subtypes of CDT (I to V) were reported in E. coli. Methods: We examined 252 E. coli strains belonging to four different groups. Of these strains, 57 were isolated from patients with diarrhea, 65 from patients with urinary tract infections (UTI), 65 from patients with sepsis and 65 from patients with other extraintestinal infections (mostly surgical wounds, decubitus ulcers and respiratory tract infections). Identification of these strains was performed by MALDI-TOF analysis and detection of genes encoding CDTs and determination of the phylogenetic group was performed by PCR. Results: In this study, we detected presence of cdt genes in 11 of 252 E. coli strains tested (4,4 %). Four cdt positive E. coli strains were confirmed in group of UTI (6,15 %), three cdt positive E. coli strains in groups of diarrhea (5,3 %) and other extraintestinal infections (4,6 %). The lowest incidence, one cdt positive E. coli strain, was observed in group of sepsis (1,5 %). All cdt positive E. coli strains belonged to phylogenetic group B2. Conclusion: CDT-producing E. coli are isolated in a low percentage from patients with intestinal and extraintestinal infections, including sepsis and our results correspond with these studies. A weak prevalence of cdt genes suggests that CDTs are not major virulence factors but in combination with other virulence factors may increase virulence potential of E. coli. We suppose that all 11 cdt positive E. coli strains represent real pathogens because they belong to the phylogenetic group B2 which is pathogenic lineage for bacteria E. coli.

Keywords: cytolethal distending toxin, E. coli, phylogenetic group, extraintestinal infection, diarrhea

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Authors: Lidia Kowalska, Edward Arseniuk

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Glume and leaf blotch is a disease of wheat caused by necrotrophic fungus Parastagonospora nodorum. It is a serious pathogen in many wheat-growing areas throughout the world. Use of resistant cultivars is the most effective and economical means to control the above-mentioned disease. Plant breeders and pathologists have worked intensively to incorporate resistance to the pathogen in new cultivars. Conventional methods of breeding for resistance can be supported by using the biotechnological ones, i.e., somatic embryogenesis and androgenesis. Therefore, an effort was undertaken to compare genetic variation in P. nodorum resistance among winter wheat somaclones, dihaploids and conventional varieties. For the purpose, a population of 16 somaclonal and 4 dihaploid wheat lines from six crosses were used to assess their resistance to P. nodorum under field conditions. Lines were grown in disease-free (fungicide protected) and inoculated micro plots in 2 replications of a split-plot design in a single environment. The plant leaves were inoculated with a mixture of P. nodorum isolates three times. Spore concentrations were adjusted to 4 x 10⁶ of viable spores per one milliliter. The disease severity was rated on a scale, where > 90% – susceptible, < 10% - resistant. Disease ratings of plant leaves showed statistically significant differences among all lines tested. Higher resistance to P. nodorum was observed more often on leaves of somaclonal lines than on dihaploid ones. On average, disease, severity reached 15% on leaves of somaclones and 30% on leaves of dihaploids. Some of the genotypes were showing low leaf infection, e.g. dihaploid D-33 (disease severity 4%) and a somaclone S-1 (disease severity 2%). The results from this study prove that dihaploid and somaclonal variation might be successfully used as an additional source of wheat resistance to the pathogen and it could be recommended to use in commercial breeding programs. The reported results prove that biotechnological methods may effectively be used in breeding for disease resistance of wheat to fungal necrotrophic pathogens.

Keywords: glume and leaf blotch, somaclonal, androgenic variation, wheat, resistance breeding

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Authors: Andreev V. A., Kovalenko T. N., Privolnev V. V., Chernavin A. V., Knyazeva E. R.

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Aim: To evaluate the antagonistic activity of Streptococcus salivarius K12 (SsK12) against ENT and oral cavity infection pathogens (S. pneumoniae, S. pyogenes, S. aureus), gram-negative bacteria (E. coli, P. aeruginosa) and C. albicans. Materials and methods: The probiotic strain SsK12 was isolated from the dietary supplement containing at least 1 × 109 CFU per tablet. The tablet was dissolved in the enrichment broth. The resulting suspension was seeded on 5% blood agar and incubated at 35°C in 4-6% CO2 for 48 hours. The raised culture was identified as Streptococcus salivarius with MALDI-TOF mass spectrometry method. The evaluation of SsK12 antagonistic activity was carried out using a perpendicular streak technique. The daily SsK12 culture was inoculated as heavy streaks with a loop at one side of Petri dish with the Muller-Hinton agar (MHA) and incubated for 24 hours at 350 C in anaerobic conditions. It was supposed that bacteriocins would diffuse over the whole area of the agar media. On the next day S. pneumoniae, S. pyogenes, S. aureus, E. coli, P. aeruginosa and C. albicans clinical isolates were streaked at the clear side of MHA Petri dish. MHA Petri dish inoculated with SsK12 (one part) and with the respective clinical isolates (another part) streaked perpendicularly on the same day was used as the control. Results: There was no growth of S. pyogenes on the Petri dish with SsK12 daily culture; the growth of a few colonies of S. pneumonia was noted. The growth of S. aureus, E. coli, P. aeruginosa and C. albicans was noted along the inoculated streak. On the control Petri dish with simultaneous inoculating of the SsK12 strain and the test cultures, the growth of all the testes isolates was noted. Conclusions: (1) SsK12 possesses perfect antagonistic activity against S. pyogenes and good activity against S. pneumoniae. (2) There was no antagonistic activity of SsK12 against S. aureus, E. coli, P. aeruginosa and C. albicans. (3) SsK12 antagonistic properties make it possible to use this probiotic strain for prophylaxis of recurrent ENT infections.

Keywords: probiotics, SsK12, streptococcus salivarius K12, antagonistic activity

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Authors: Pampita Chakraborty, Sukumar Mukherjee

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This study was done to determine the prevalence of nosocomial infections in the ICU and to identify the common microorganisms causing these infections and their antimicrobial sensitivity pattern. Nosocomial infection or hospital-acquired infection is a localized or a systemic condition resulting from an adverse reaction to the presence of infectious agents. Nosocomial infections are not present or incubating when the patient is admitted to hospital or other health care facility. They are caused by pathogens that easily spread through the body. Many hospitalized patients have compromised immune systems, so they are less able to fight off infections. These infections occur worldwide, both in the developed and developing the world. They are a significant burden to patients and public health. They are a major cause of death and increased morbidity in hospitalized patients, which is a matter of serious concern today. This study was done during the period of one year (2012-2013) in the ICU of the tertiary care hospital in eastern India. Prevalence of nosocomial infection was determined; site of infection and the pattern of microorganisms were identified along with the assessment of antibiotic susceptibility profile. Patients who developed an infection after 48 hours of admission to the ICU were included in the study. A total of 324 ICU patients were analyzed, of these 79 patients were found to have developed a nosocomial infection (24.3% prevalence). Urinary tract infection was found to be more predominant followed by respiratory tract infection and soft tissue infection. The most frequently isolated microorganism was E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae followed by other organisms respectively. Antibiotic susceptibility test of these isolates was done against commonly used antibiotics. Patients admitted to the ICU are especially susceptible to nosocomial infections. Despite adequate antimicrobial treatment, nosocomial ICU infections can significantly affect ICU stay and can cause an increase in patient’s morbidity and mortality. Adherence to infection protocol, proper monitoring and the judicious use of antibiotics are important in preventing such infections on a regular basis.

Keywords: antibiotic susceptibility, intensive care unit, nosocomial infection, nosocomial pathogen

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Authors: Mohammadreza Hajialigol, Nargues Falahi Charkhabi, Fatemeh Shahryari, Saadat Sarikhani

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BACKGROUND AND OBJECTIVES Iran is the third producer of Persian walnut worldwide. However, its walnut trees have been under threat from decline during last decade. Walnut canker caused by B. nigrifluens and B. rubrifaciens was recorded in multiple regions of Iran. Furthermore, Brenneria rosae subsp. rosae and Gibbsiella quercinecans were recently recognized as responsible for walnut decline in northwestern Iran. This study aimed to identify the causal agent of walnut decline in Kermanshah and Isfahan. MATERIAL AND METHODS Symptomatic samples were collected from affected walnut trees of Kermanshah and Isfahan provinces. The pathogenicity of strains was proved on immature walnut fruits cv. ‘Hartley’ and young green twigs of two-year-old walnut seedling cv. ‘Chandler’. Pathogenic strains were subjected to conventional phenotypic tests. 16S rRNA, gyrB, and infB genes were partially amplified and sequenced. RESULTS Irregular longitudinal cankers and dark lesions were observed in the outer and inner bark, respectively. Twenty-four strains were isolated on EMB-agar media. Fourteen strains were able to cause necrosis and a dark-colored region in the mesocarp and on young green twigs around the inoculation site 14 and 30 days post-inoculation, respectively. Strains were able to hydrolyze Tween 20, Tween 80, gelatin and esculin, however, did not produce indole or urease. Pairwise comparison, the 16S rRNA gene nucleotide sequences of strain I2 were 100% identical with those of Rahnella victoriana FRB 225T. Moreover, a phylogenetic tree reconstructed based on the concatenated sequences of two housekeeping gene fragments, gyrB (601 bp) and infB (615 bp), revealed that the strains I2, I5, and KE6 were clustered with R. victoriana FRB 225T. CONCLUSION To the best of our knowledge, this is the first report of R. victoriana in association with walnut decline. This result is necessary to find resistant genotypes.

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: V. Konjik, J. Schwartz, R. Sandhoff, M. Mack

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The rising number of multi-resistant pathogens demands the development of new antibiotics in order to reduce the lethal risk of infections. Here, we investigate roseoflavin, a vitamin B2 analogue which is produced by Streptomyces davawensis and Streptomyces cinnabarinus. We consider roseoflavin to be a 'Trojan horse' compound. Its chemical structure is very similar to riboflavin but in fact it is a toxin. Furthermore, it is a clever strategy with regard to the delivery of an antibiotic to its site of action but also with regard to the production of this chemical: The producer cell has only to convert a vitamin (which is already present in the cytoplasm) into a vitamin analog. Roseoflavin inhibits the activity of Flavin depending proteins, which makes up to 3.5 % of predicted proteins in organisms sequenced so far. We sequentially knocked out gene clusters and later on single genes in order to find the ones which are involved in the roseoflavin biosynthesis. Consequently, we identified the gene rosB, coding for the protein carrying out the first step of roseoflavin biosynthesis, starting form Flavin mononucleotide. Here we show, that the protein RosB has so far unknown features. It is per se an oxidoreductase, a decarboxylase and an aminotransferase, all rolled into one enzyme. A screen of cofactors revealed needs of oxygen, NAD+, thiamine and glutamic acid to carry out its function. Surprisingly, thiamine is not only needed for the decaboxylation step, but also for the oxidation of 8-demethyl-8-formyl Flavin mononucleotide. We had managed to isolate three different Flavin intermediates with different oxidation states, which gave us a mechanistic insight of RosB functionality. Our work points to a so far new function of thiamine in Streptomyces davawensis. Additionally, RosB could be extremely useful for chemical synthesis. Careful engineering of RosB may allow the site-specific replacement of methyl groups by amino groups in polyaromatic compounds of commercial interest. Finally, the complete clarification of the roseoflavin biosynthesis opens the possibility of engineering cost-effective roseoflavin producing strains.

Keywords: antibiotic, flavin analogue, roseoflavin biosynthesis, vitamin B2

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Authors: Mohammadreza Hajialigol, Nargues Falahi Charkhabi, Fatemeh Shahryari, Saadat Sarikhani

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BACKGROUND AND OBJECTIVES Walnut canker is characterized by brown to blackish roundish blotches on the trunks and main branches, necrosis of inner bark and bleeding with dark brown to black-colored exudates. The present study aimed to identify the causative agents of walnut decline by their phenotypic features, approval of pathogenicity, the partial sequencing of the housekeeping genes in Razavi Khorasan. MATERIAL AND METHODS Ten Symptomatic samples were collected from walnut orchards of Razavi Khorasan in 2019. Pathogenicity of all isolated strains was carried out on walnut immature fruits cv. ‘Hartley’ and young green twigs of cv. ‘Chandler’. All pathogenic strains were subjected to physiological, morphological and biochemical tests. 16S rRNA and housekeeping genes (fusA, leuS, and pyrG) were partially amplified and sequenced. RESULTS Eight strains were able to cause necrosis and a dark-colored region in the mesocarp of immature walnut fruits, and three representative strains caused necrosis on young inoculated twigs. Strains utilized starch, however, did not utilized esculin, Tween 20, Tween 80, and gelatin. The partial 16S rRNA gene sequence of strain KH7 indicated 99.63 % similarity to that of Citrobacter braakii ATCC5113T. The phylogenetic analyses based on the partial sequencing of three housekeeping genes, fusA (633 bp), pyrG (305), and leuS (640 bp), demonstrated that strains KH1, KH3, and KH7 belong to C. braakii species in a monophyletic clade with high bootstrap support. CONCLUSION To the best of our knowledge, this is the first report of C. braakii as a new plant pathogen which cause walnut decline. Identification of bacteria associated with walnut decline will eventually improve our understanding of the etiology of the disease and may result in improved management techniques for control.

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: Hardik Goswami, Gayatri Swarnakar

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-Mastitis disease has been known as one of the most costly diseases of dairy cattle and observed as an inflammatory disease of cow and buffalo udder. Mastitis badly affected animal health, quality of milk and economics of milk production along with cause’s great economic loss. Bacteria have been representing the most common etiological agents of mastitis. The antibiotic sensitivity test was important to attain accurate treatment of mastitis. The aim of present research work was to explore prevalence and antibiotic susceptibility pattern of bacterial isolates recovered from cow and buffalo clinical mastitis milk sample. During the period of April 2010 to April 2014, total 1487 clinical mastitis milk samples of cow and buffalo were tested to check the prevalence of mastitis causing bacterial isolates. Milk samples were collected aseptically from the udder at the time of morning milking. The most prevalent bacterial isolates were Staphylococcus aureus (24.34%) followed by coliform bacteria (15.87%), coagulase negative Staphylococcus aureus (13.85%), non-coliform bacteria (13.05%), mixed infection (12.51%), Streptococcus spp. (10.96%). Out of 1487, 140 (9.42%) mastitis milk samples showed no growth on culture media. Identification of bacteria made on the basis of Standard Microbial features and procedures. Antibiotic susceptibility of bacterial isolates was investigated by Kirby-Bauer disk diffusion method. In vitro Antibiotic susceptibility test of bacterial isolates revealed higher sensitivity to Gentamicin (74.6%), Ciprofloxacin (62.1%) and Amikacin (59.4%). The lower susceptibility was shown to Amoxicillin (21.6%), Erythromycin (26.4%) and Ceftizoxime (29.9%). Antibiotic sensitivity pattern revealed Gentamicin are the possible effective antibiotic against the major prevalent mastitis pathogens. Present research work would be helpful in increase production, quality and quantity of milk, increase annual income of dairy owners and improve health of cow and buffaloes.

Keywords: antibiotic, buffalo, cow, mastitis, prevalence

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Authors: Lubna Azmi, Ila Shukla, Shyam Sundar Gupta, Padam Kant, Ch. V. Rao

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Gastric ulcer is a major global health threat, and it is the leading cause of stomach cancer death worldwide. Helicobacter pylori bacteriumis the most important etiologic factor for gastric ulcer. This infection is highly pervasive in South Asian developing countries, especially in India, Nepal, Srilanka etc. due to diversification in geographic area. Pathophysiology of gastric mucosal damage associated with non-invasive bacterium has not justified in detail, but it leads to change in histopathology, immunochemistry of the gastric and duodenal reason of host. The mechanism responsible for bacteria tissue tropism and mucosal damage in stomach variance during the disease is not clearly described and understood scientifically in treatment and control of pathogenic organisms. Polyphenols are secondary metabolites of plants and are generally involved in defense against aggression by pathogens. 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one and 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde are polyphenolic compound obtained from popular Indian medicinal plants ghavpatta (ArgeriaspeciosaLinn.f) andBael (Aeglemarmelos) have long been used in traditional Ayurvedic Indian medicine for various diseases. They have promising effects on ulcer, as detailed investigation has made in our laboratory. Therefore, the aim of present study is to explore membrane –dependent morphogenesis of H. pylori and associated apoptosis-mediated cell death. Based on this we analyzed immune gene expression in stomach of experimental animals with H. pylori, using quantitative reverse transcription polymerase chain reaction(q RT-PCR). This revealed rapid induction of prostaglandin, interferon I (INF-I), interferon II (INF-II) and INF-I associated genes in the infected animal. Ultrastructural changes associated with H. pylori will be taken for advanced studies. This investigation shows that the biomarkers eradicate H. pylori bacterium caused gastric ulcer which is a major risk factor for gastric cancer.

Keywords: gastric ulcer, Helicobacter pylori, immunochemistry, polyphenols

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Authors: Raana Babadi Fathipour

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The production of dairy holds significant importance in the sustenance of billions of individuals worldwide, as they rely on milk and its derived products for daily consumption. In addition to being a source of essential nutrients crucial for human well-being, such as proteins, fats, vitamins, and minerals; dairy items are witnessing an increasing demand worldwide. Amongst all the factors contributing to the quality and safety assurance of dairy products, the strong focus lies on maintaining high standards in raw milk procurement. Raw milk serves as an externally nutritious medium for various microorganisms due to its inherent properties. This poses a considerable challenge for the dairy industry in ensuring that microbial contamination is minimized throughout every stage of the value chain. Despite implementing diverse process technologies—both conventional and innovative—the occurrence of microbial spoilage still results in substantial losses within this industry context. Moreover, milk and dairy products have been associated with numerous cases of foodborne illnesses across the globe. Various pathogens such as Salmonella serovars, Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and enterotoxin producing Staphylococcus aureus are commonly identified as the culprits behind these outbreaks in the dairy industry. The effective management of food safety within this sector necessitates a proactive and risk-based approach to reform. However, this strategy presents difficulties for developing nations where informal value chains dominate the dairy sector. Whether operating on a small or large scale or falling within formal or informal realms, it is imperative that the dairy industry adheres to principles of good hygiene practices and good manufacturing practices. Additionally, identifying and managing potential sources of contamination is crucial in mitigating challenges pertaining to quality and safety precautions.

Keywords: dairy value chain, microbial contamination, food safety, hygiene

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Authors: Neslihan Ozsoy Taskiran, Miray Dayioglu, Belgin Gunbey, Banu Yucel, Cigdem Takma, Unal Karik, Tugce Olgun, Levent Aydin

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A greater wax moth (Galleria mellonella L.), which is one of the most important pests after Varroa, plays a role in the transportation of many pathogens into the hive as well as damage to the honeycombs, and beekeepers suffer economically. Due to the risk that some of the methods against this pest may cause residue in bee products, and it can be harmful to the health of people who consume these products. Therefore, the most appropriate, most economical, and effective method should be applied in the moth control. For this purpose, in the first phase of the project (2017-2018), planned to be 2-stage in the Aegean Agricultural Research Institute in 2017-2020, the honeycombs, certified with good agricultural practice, were kept in a favorable condition for moths. Later, applications (Sulfur - B401 - Walnut (Leaf & Smoker) - lavender essential oil (1cc & 2cc & 3cc & 4cc) - laurel essential oil (1cc & 2cc & 3cc & 4cc) - control) were applied to the honeycombs with moths. In 2017, the B401 group had the highest wax moth damage area, and the group with the lowest wax moth damage area was determined as lavender 1cc; In 2018, the highest wax moth damage area was found in the walnut smoker group, while the lowest wax moth damage area was found in sulfur, walnut leaves, laurel 1cc - 2cc - 4cc, lavender 1cc - 2cc - 3cc - 4cc and control groups. In addition, sulfur residue amount (mean 128,18 mg/kg) in honeycomb was measured in the sulfur-treated group. Phase 1 of the project was completed, and the most important sub-groups among walnut (leaf) - lavender (1cc) and laurel (4cc) groups were identified. Accordingly, it is planned to carry out these treatments ((sulfur - B401 - walnut (leaf) - lavender (1cc) and laurel (4cc)) on honeycombs with do not contain moths, and later, it is planned to examine the effects of the treatment on the offspring area and honey yield by giving these honeycombs to the hives, in the 2nd stage of the project (2019-2020).

Keywords: honey bee, lavender essential oil, laurel essential oil, walnut, wax moth

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Authors: Umar Rehman, Holly Roy, K. T. Tsang, D. S. Jeyaretna, W Singleton, B. Fisher, P. A. Glew, J. Greig, Peter C. Whitfield

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Introduction: A brain abscess is a life-threatening condition, carrying significant mortality. It requires rapid identification and treatment. Management involves a combination of antibiotics and surgery. The aim of the current study was to identify common bacteria responsible for cerebral abscesses as well as the long term functional and neurological outcomes of patients following treatment in a retrospective series at a single UK neurosurgical centre. Methodology: We analysed patients that had received a diagnosis of 'cerebral abscess' or 'subdural empyema' between June 2002 and June 2018. This was done in the form of a retrospective review. The search resulted in a total of 180 patients; with 37 patients being excluded (spinal abscess, below 18 or non-abscess related admissions). Data were collected from medical case notes including information about demographics, comorbidities, immunosuppression, presentation, size/location of lesions, pathogens, treatment, and outcomes. Results: In total, we analysed 143 patients between the ages of 18-90. Focal neurological deficit and headaches were seen in 84% and 68% of patients respectively. 108 positive brain cultures were seen; with the largest proportion, 59.2% being gram-positive cocci, with strep intermedius being the most common pathogen identified in 13.9% of patients. Of the patients with positive blood cultures (n=11), 72.7% showed the same organism both in the blood and on the brain cultures. Long term outcomes (n=72) revealed that 48% of patients seizure-free without requiring anti-epileptics, 51.3% of patients had full recovery of their neurological symptoms. There was a mortality rate of 13.9% in the series. Conclusion: In conclusion, the largest bacterial cause of abscess within our population was due to gram-positive cocci. The majority of the patient demonstrated full neurological recovery with close to half of patients not requiring anti-epileptics following discharge.

Keywords: bacteria, cerebral abscess, long term outcome, neurological deficit

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Authors: Alma Ramić, Mirjana Skočibušić, Renata Odžak, Tomica Hrenar, Ines Primožič

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In an attempt to identify a new class of antimicrobial agents, the antimicrobial potential of Cinchona alkaloid derivatives was evaluated. The bark of the Cinchona trees is the source of a variety of alkaloids, among which the best known are quinine, quinidine, cinchonine and cinchonidine. They are very useful as organocatalysts in stereoselective synthesis. On the other hand, quinine is traditionally used in the treatment of malaria. Furthermore, Cinchona alkaloids possess various analgesic, anti-inflammatory and anti–arrhythmic properties as well. In this work we present the synthesis of twenty quaternary derivatives of pseudo−enantiomeric Cinchona alkaloid derivatives to evaluate their antibacterial activity. Quaternization of quinuclidine moiety was carried out with groups diverse in their size. The structures of compounds were systematically modified to obtain drug-like properties with proper physical and chemical properties and avoiding toxophore. All compounds were prepared in good yields and were characterized by standard analytical spectroscopy methods (1D and 2D NMR, IR, MS). The antibacterial activities of all compounds were evaluated against series of recent clinical isolates of antibiotic susceptible Gram-positive and resistant Gram-negative pathogens by determining their zone of inhibition and minimum inhibitory concentrations. All compounds showed good to strong broad-spectrum activity, equivalent or better in comparison with standard antibiotics used. Furthermore, seven compounds exhibited significant antibacterial efficiency against Gram-negative isolates. To visualize the results, principal component analysis was used as an additional classification tool. Cytotoxicity of compounds with different cell lines in human cell culture was determined. Based on these results, substituted quaternary Cinchona scaffold can be considered as promising new class of antimicrobials and further investigations should be performed. Supported by Croatian Science Foundation, Project No 3775 ADESIRE.

Keywords: antibacterial efficiency, cinchona alkaloids, cytotoxicity, pseudo‐enantiomers

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Authors: Birhane Atnafu, Chemeda Abedeta Garbaba, Fikre Lemessa, Abdi Mohammed, Alemayehu Chala

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Essential oil is a bio-pesticide plant product used as an alternative to pesticides in managing plant pests, including fungal pathogens. Thus, the current study aims to investigate the chemical composition and antifungal activities of essential oils (EO) extracted from three aromatic plants i.e., Thymus vulgaris, Coriandrum sativum, and Cymbopogon martini. The leaf parts of those selected plants were collected from the Jimma area and their essential oil was extracted by hydro-distillation method in a Clevenger apparatus. The chemical composition of selected plant essential oil was analyzed by using Gas chromatography-mass spectrometry (GC/MS) and their inhibitory effects were tested in vitro on toxigenic fungi isolated from maize kernel. Chemical analysis results revealed the presence of 32 compounds in C. sativum with Hexanedioic acid, bis (2-ethylhexyl) ester (46. 9%), 2-Decenal, (E)- (12.6), and linalool (8.3%) being the dominant ones. T. vulgaris essential oils constituted 25 compounds, of which thymol (34.4%), o-cymene (17.5%), and Gamma-Terpinene (16.8%) were the major components. Twenty-five compounds were detected in C. martinii of which geraniol (51.4%), Geranyl acetate (14.5%), and Trans – ß-Ocimene (11.7%) were dominant. The EOs of the tested plants had very high antifungal activity (up to 100% efficacy) against Aspergillus flavus, Aspergillus niger, Fusarium graminearum and Fusarium verticillioides in vitro and on maize grains. The antifungal activities of these essential oils were dependent on the major components such as thymol, hexanedioic acid, bis (2-ethylhexyl) ester, and geraniol. The study affirmed the potential of these essential oils controlling as bio-fungicides to manage the effects of potentially toxigenic fungi associated with maize under post-harvest stages. This can reduce the consequences of the health impacts of the mold and toxigenic compounds produced in maize.

Keywords: bio-activity, bio-pesticides, maize, mycotoxin

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Authors: P. Efstathiou, E. Kouskouni, Z. Manolidou, K. Karageorgou, M. Tseroni, A. Efstathiou, V. Karyoti, I. Agrafa

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Aim: The aim of this prospective comparative study is to evaluate the reduction of microbial flora on the surface of an axillary digital thermometer, made of antimicrobial copper, in relation with a common digital thermometer. Material – Methods: A brand new digital electronic thermometer implemented with antimicrobial copper (Cu 70% - Nic 30%, low lead) on the two edges of the device (top and bottom: World Patent Number WO2013064847 and Register Number by the Hellenic Copper Development Institute No 11/2012) was manufactured and a comparative study with common digital electronic thermometer was conducted on 18 ICU (Intensive Care Unit) patients of three different hospitals. The thermometry was performed in accordance with the projected International Nursing Protocols for body temperature measurement. A total of 216 microbiological samples were taken from the axillary area of the patients, using both of the investigated body temperature devises. Simultaneously the “Halo” phenomenon (phenomenon “Stefanis”) was studied at the non-antimicrobial copper-implemented parts of the antimicrobial digital electronic thermometer. Results: In all samples collected from the surface of the antimicrobial electronic digital thermometer, the reduction of microbial flora (Klebsiella spp, Staphylococcus aureus, Staphylococcus epidermitis, Candida spp, Pneudomonas spp) was progressively reduced to 99% in two hours after the thermometry. The above flora was found in the axillary cavity remained the same in common thermometer. The statistical analysis (SPSS 21) showed a statistically significant reduction of the microbial load (N = 216, < 0.05). Conclusions: The hospital-acquired infections are linked to the transfer of pathogens due to the multi-usage of medical devices from both health professionals and patients, such as axillary thermometers. The use of antimicrobial digital electronic thermometer minimizes microbes' transportation between patients and health professionals while having all the conditions of reliability, proper functioning, security, ease of use and reduced cost.

Keywords: antimicrobial copper, cross infections, digital thermometers, ICU

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Authors: Huyen T. Pham, Nhue P. Nguyen, Tien Q. Phi, Phuong T. Dang, Hy G. Le

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Since the marine environmental conditions are extremely different from the other ones, so that marine actinomycetes might produce novel bioactive compounds. Therefore, actinomycete strains were screened from marine water and sediment samples collected from the coastal areas of Northern Vietnam. Ninety-nine actinomycete strains were obtained on starch-casein agar media by dilution technique, only seven strains, named HP112, HP12, HP411, HPN11, HP 11, HPT13 and HPX12, showed significant antibacterial activity against both gram-positive and gram-negative bacteria (Bacillus subtilis ATCC 6633, Staphylococcus epidemidis ATCC 12228, Escherichia coli ATCC 11105). Further studies were carried out with the most active HP411strain against Candida albicans ATCC 10231. This strain could grow rapidly on starch casein agar and other media with high salt containing 7-10% NaCl at 28-30oC. Spore-chain of HP411 showed an elongated and circular shape with 10 to 30 spores/chain. Identification of the strain was carried out by employing the taxonomical studies including the 16S rRNA sequence. Based on phylogenetic and phenotypic evidence it is proposed that HP411 to be belongs to species Streptomyces variabilis. The potent of the crude extract of fermentation broth of HP411that are effective against wide range of pathogens: both gram-positive, gram-negative and fungi. Further studies revealed that the crude extract HP411 could obtain the anticancer activity for cancer cell lines: Hep-G2 (liver cancer cell line); RD (cardiac and skeletal muscle letters cell line); FL (membrane of the uterus cancer cell line). However, the actinomycetes from marine ecosystem will be useful for the discovery of new drugs in the furture.

Keywords: marine actinomycetes, antibacterial, anticancer, Streptomyces variabilis

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Authors: Nemat Sokhandan Bashir

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Threats from viruses to agricultural crops could be even larger than the losses caused by the other pathogens because, in many cases, the viral infection is latent but crucial from an epidemic point of view. Wild vegetation can be a source of many viruses that eventually find their destiny in crop plants. Although often asymptomatic in wild plants due to adaptation, they can potentially cause serious losses in crops. Therefore, exploring viruses in wild vegetation is very important. Recently, omics have been quite useful for exploring plant viruses from various plant sources, especially wild vegetation. For instance, we have discovered viruses such as Ambrossia asymptomatic virus I (AAV-1) through the application of metagenomics from Oklahoma Prairie Reserve. Accordingly, extracts from randomly-sampled plants are subjected to high speed and ultracentrifugation to separated virus-like particles (VLP), then nucleic acids in the form of DNA or RNA are extracted from such VLPs by treatment with phenol—chloroform and subsequent precipitation by ethanol. The nucleic acid preparations are separately treated with RNAse or DNAse in order to determine the genome component of VLPs. In the case of RNAs, the complementary cDNAs are synthesized before submitting to DNA sequencing. However, for VLPs with DNA contents, the procedure would be relatively straightforward without making cDNA. Because the length of the nucleic acid content of VPLs can be different, various strategies are employed to achieve sequencing. Techniques similar to so-called "chromosome walking" may be used to achieve sequences of long segments. When the nucleotide sequence data were obtained, they were subjected to BLAST analysis to determine the most related previously reported virus sequences. In one case, we determined that the novel virus was AAV-l because the sequence comparison and analysis revealed that the reads were the closest to the Indian citrus ringspot virus (ICRSV). AAV—l had an RNA genome with 7408 nucleotides in length and contained six open reading frames (ORFs). Based on phylogenies inferred from the replicase and coat protein ORFs of the virus, it was placed in the genus Mandarivirus.

Keywords: wild, plant, novel, metagenomics

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Authors: Recep Keşli, Gülşah Aşık, Cengiz Demir, Onur Türkyılmaz

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Objective: Proteus mirabilis (P. mirabilis) is one of Gram-negative pathogens in human and it causes urinary tract and nosocomial infections. P. mirabilis is susceptible to β-lactams, aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. It was aimed to investigate the resistance status to antimicrobial agents of Proteus mirabilis strains produced from samples sent to Afyon Kocatepe University, ANS Research and Practice Hospital, Microbiology Laboratory from different clinics and polyclinics during the period of 24 months. Methods: Between October 2013 and September 2015, a total of 30 Proteus were isolated from clinical samples of patients were hospitalized in intensive care units and in various departments of Afyon Kocatepe University, ANS Research and Practice Hospital. Identification of the bacteria was determined by conventional methods and VITEK 2 system (bioMérieux, France) was used additionally. Antibacterial susceptibility tests were performed by Kirby Bauer disc (Oxoid, Hempshire, England) diffusion method following the recommendations of CLSI. Results: Of the total 30 Proteus strains isolated from clinical samples, 19 from urine, 7 from wound, 4 from tracheal aspiration materials were isolated. Antimicrobial resistant for these strains were determined to 24,3% for meropenem, 26.2% for imipenem, 20.2% for amikacin 10.5% for cefepim, 33.3% for ciprofloxacin and levofloxacine, 31.6% for ceftazidime, 20% for ceftriaxone, 15.2% for gentamicin and 26.6% for amoxicillin-clavulanate, 26.2% trimethoprim-sulfamethoxale. Conclusion: In the present study, the highest number of clinical isolates of P. mirabilis were isolated from urine (63,3%), followed by the others (36,6%). The distribution of samples P. mirabilis strains to the clinics were as fallows; 16,8% intensive care unit (ICU), 29,9% polyclinics, 53,3% hospital service units The most effective antibiotic on the total of strains were found to be cefepim, the least effective antibiotics on the total of strains were found to be trimethoprim-sulfamethoxale.

Keywords: proteus mirabilis, antibiotic resistance, intensive care unit, Proteus spp.

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Authors: Haftay Abraha Tadesse, Atsebaha Gebrekidan Kahsay, Mahumd Abdulkader

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Background: Salmonella species and Escherichia coli are important foodborne pathogens affecting humans and animals. They are among the most important causes of infection that are associated with the consumption of contaminated food. This study was aimed to determine the prevalence, antimicrobial susceptibility patterns and associated risk factors for Salmonella species and E. coli in raw meat from butchery houses of Mekelle, Northern Ethiopia. Methodology: A cross-sectional study was conducted from January to September 2019. Socio-demographic data and risk factors were collected using a predesigned questionnaire. Meat samples were collected aseptically from the butchery houses and transported using icebox to Mekelle University, College of Veterinary Sciences for the isolation and identification of Salmonella species and E. coli, Antimicrobial susceptibility patterns were determined using Kirby disc diffusion method. Data obtained were cleaned and entered into Statistical Package for the Social Sciences version 22 and logistic regression models with odds ratio were calculated. P-value < 0.05 was considered as statistically significant. Results: A total of 153 out of 384 (39.8%) of the meat specimens were found to be contaminated. The contamination of Salmonella species and E. coli were 15.6% (n=60) and 20.8%) (n=80), respectively. Mixed contamination (Salmonella species and E. coli) was observed in 13 (3.4 %) of the analyzed. Poor washing hands regularly (AOR = 8.37; 95% CI: 2.75-25.50) and not using gloves during meat handling (AOR=11. 28; 95% CI: (4.69 27.10) were associated with an overall bacterial contamination.About 95.5% of the tested isolates were sensitive to chloramphenicol and norfloxacin while the resistance of amoxyclav_amoxicillin and erythromycin were both isolated bacteria species. The overall multidrug resistance pattern for Salmonella and E. coli were 51.4% (n=19) and 31.8% (14), respectively. Conclusion: Of the 153 (153/384) contaminated raw meat, 60 (15.6%) and 80 (20.8%) were contaminated by Salmonella species and E. coli, respectively. Poor hand washing practice and not using glove during meat handling showed significant association with bacterial contamination. Multidrug-resistant showed in Salmonella species and E. coli were 19 (51.4%) and 14 (31.8%), respectively.

Keywords: antimicrobial susceptibility test, butchery houses, e. coli, salmonella species

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Authors: S. Ben-Shabat, T. Turgeman, O. Leubinski, N. Roth-Bejerano, V. Kagan-Zur, Y. Sitrit

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The ectomycorrhizal (ECM) desert truffle Terfezia boudieri produces edible fruit bodies and forms symbiosis with its host plant Helianthemum sessiliflorum (Cistaceae) in the Negev desert of Israel. The symbiosis is vital for both partners' survival under desert conditions. Under desert habitat conditions, ECMs must form symbiosis before entering the dry season. To secure a successful encounter, in the course of evolution, both partners have responded by evolving special signals exchange that facilitates recognition. Members of the Cistaceae family serve as host plants for many important truffles. Conceivably, during evolution a common molecule present in Cistaceae plants was recruited to facilitate successful encounter with ectomycorrhizas. Arbuscular vesicular fungi (AM) are promiscuous in host preferences, in contrast, ECM fungi show specificity to host plants. Accordingly, we hypothesize that H. sessiliflorum secretes a chemotrophic-signaling, which is common to plants hosting ECM fungi belonging to the Pezizales. However, thus far no signaling molecules have been identified in ECM fungi. We developed a bioassay for chemotrophic activity. Fractionation of root exudates revealed a substance with chemotrophic activity and molecular mass of 534. Following the above concept, screening the transcriptome of Terfezia, grown under chemoattraction, discovered genes showing high homology to G proteins-coupled receptors of plant pathogens involved in positive chemotaxis and chemotaxis suppression. This study aimed to identify the active molecule using analytical methods (LC-MS, NMR etc.). This should contribute to our understanding of how ECM fungi communicate with their hosts in the rhizosphere. In line with the ability of Terfezia to form also endomycorrhizal symbiosis like AM fungi, analysis of the mechanisms may likewise be applicable to AM fungi. Developing methods to manipulate fungal growth by the chemoattractant can open new ways to improve inoculation of plants.

Keywords: chemotrophic signal, Helianthemum sessiliflorum, Terfezia boudieri, ECM

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Authors: Hafiza Javaria Ashraf, Asim Abbasi, Muhammad Hussnain Babar, Muhammad Sufyan

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Brevicoryne brassicae (Aphid) is not only the major biotic constraint of rapeseed crop but also transmits 20 different viral pathogens that cause diseases in crucifers. Aphids cause major losses to rapeseed by stunting growth and yield, with real damage being contamination of harvested heads. The misuse of pesticides has led to tremendous economic losses and hazards to human health and environmental pollution. Thus, newer approaches for pest control are continuously being sought. The naturally occurring, biologically active plant-based products seem to have a prominent role in the development of future commercial pesticides not only for increased productivity but their eco-friendly nature. The present experiment was carried out in Research Area of Ayub Agriculture Research Institute, Faisalabad to check the efficacy of different botanicals against rapeseed aphid. The tested botanicals were, neem seed extract, neem leaf extract, dathora seed extract, kaner leaf extract and aak leaf extract. Insecticide, advantage 20 EC served as the positive control in the experiment. Data was recorded before and after 1, 3 and 7 days of treatment application. The results of the experiment revealed that neem seed extract exhibited maximum mortality (48.42%) followed by dathora (45.54%) and kaner leaf extract (40.29%) after 7 days of treatment application. However minimum mortality i.e. 26.64% was observed in case of aak leaf extract. Advantage encountered maximum mortality i.e. 86.14%. All treatments caused maximum mortality after 7 days of treatment application. In case of pollinators maximum population reduction was observed in case of insecticide (74.29%) while minimum reduction was observed in neem leaf extract (11.57%). Hence it was concluded that unlike insecticides, plant based products can be a better option for regulating pests and conserving beneficial insect fauna.

Keywords: Aphid, mortality, plant based, pollinators

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Authors: Miroslava Markovic, Snezana Rajkovic, Ljubinko Rakonjac, Aleksandar Lucic

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Oak powdery mildew is a serious problem on seedlings in nurseries as well as on naturally and artificially introduced progeny. The experiments were set on oak seedlings in two nurseries located in Central Serbia, where control of oak powdery mildew Microsphaera alphitoides Griff. et Maubl. had been conducted through alternative protection measures by means of various dosages of AQ-10 biofungicide, with and without added polymer (which has so far never been used in this country for control of oak powdery mildew). Simultaneous testing was conducted on the efficiency of a chemical sulphur-based preparation (used in this area for many years as a measure of suppression of powdery mildews, without the possibility of developing resistance of the pathogen to the active matter). To date, the Republic of Serbia has registered no fungicides for suppression of pathogens in the forest ecosystems. In order to introduce proper use of new disease-fighting agents into a country, certain relevant principles, requirements and criteria prescribed by the Forest Stewardship Council (FSC) must be observed, primarily with respect to measures of assessment and mitigation of risks, the list of dangerous and highly dangerous pesticides with the possibility of alternative protection. One of the main goals of the research was adjustment of the protective measures to the FSC policy through selection of eco-toxicologically favourable fungicides, given the fact that only preparations named on the list of permitted active matters are approved for use in certified forests. The results of the research have demonstrated that AQ-10 biofungicide can be used as a part of integrated disease management programmes as an alternative, through application of several treatments during vegetation and combination with other active matters registered for these purposes, so as to curtail the use of standard fungicides for control of powdery mildews on oak seedlings in nurseries. The best results in suppression of oak powdery mildew were attained through use of AQ-10 biofungicide (dose 50 or 70g/ha) with added polymer Nu Film-17 (dose 1.0 or 1.5 l/ha). If the treatment is applied at the appropriate time, even fewer number of treatments and smaller doses will be just as efficient.

Keywords: oak powdery mildew, biofungicides, polymers, Microsphaera alphitoides

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Authors: Jun Hyung Lee, Robin B. Guevarra, Jin Ho Cho, Bo-Ra Kim, Jiwon Shin, Doo Wan Kim, Young Hwa Kim, Minho Song, Hyeun Bum Kim

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Salmonella is one of the most important swine pathogens, causing acute or chronic digestive diseases, such as enteritis. The acute form of enteritis is common in young pigs of 2-4 months of age. Salmonellosis in swine causes a huge economic burden to swine industry by reducing production. Therefore, it is necessary that swine industries should strive to decrease Salmonellosis in pigs in order to reduce economic losses. Thus, we tested three types of natural plant extracts(PEs) to evaluate antibacterial effects against Salmonella enterica Typhimurium isolated from the piglet with Salmonellosis. Three PEs including turmeric oleoresin (containing curcumin 79 to 85%), capsicum oleoresin (containing capsaicin 40%-40.1%), and garlic essential oil (100% natural garlic) were tested using the direct contact agar diffusion test, minimum inhibitory concentration test, growth curve assay, and heat stability test. The tests were conducted with PEs at each concentration of 2.5%, 5%, and 10%. For the heat stability test, PEs with 10% concentration were incubated at each 4, 20, 40, 60, 80, and 100 °C for 1 hour; then the direct contact agar diffusion test was used. For the positive and negative controls, 0.5N HCl and 1XPBS were used. All the experiments were duplicated. In the direct contact agar diffusion test, garlic essential oil with 2.5%, 5%, and 10% concentration showed inhibit zones of 1.5cm, 2.7cm, and 2.8cm diameters compared to that of 3.5cm diameter for 0.5N HCl. The minimum inhibited concentration of garlic essential oil was 2.5%. Growth curve assay showed that the garlic essential oil was able to inhibit Salmonella growth significantly after 4hours. The garlic essential oil retained the ability to inhibit Salmonella growth after heat treatment at each temperature. However, turmeric and capsicum oleoresins were not able to significantly inhibit Salmonella growth by all the tests. Even though further in-vivo tests will be needed to verify effects of garlic essential oil for the Salmonellosis prevention for piglets, our results showed that the garlic essential oil could be used as a potential natural agent to prevent Salmonellosis in swine.

Keywords: garlic essential oil, pig, salmonellosis, Salmonella enterica

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Authors: Stéfani T. A. Dantas, Laura T. S. Takume, Bruna F. Rossi, Érika R. Bonsaglia, Ivana G. Castilho, José C. F. Pantoja, Ary Fernandes Júnior, Juliano L. Gonçalves, Marcos V. Santos, Rinaldo A. Mota, Vera L. M. Rall

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Staphylococcus aureus is one of the main pathogens causing contagious bovine mastitis, being more frequently isolated from subclinical form, although the clinical form also occurs. Clinical mastitis cause visual signs, such as swelling, fever, hardening of the mammary gland, or any change in the characteristics of the milk. Considering the subclinical type, there are no visible signs in the animal nor changes in the milk. S. aureus has many important virulence factors for the establishment of its pathogenicity in animals, such as enterotoxins, which are also responsible for foodborne poisoning. Our objective is to perform a comparative analysis between 103 isolates of S. aureus, obtained from the milk of cows with clinical mastitis and 103 more, from subclinical type, in relation to the presence of these enterotoxins and verify if their presence plays an important role in the signs of illness. We will investigate all enterotoxins described till now, such as sea-see, seg-sez, sel26, sel 27, se01, and se02 (This study was approved by the Sao Paulo State University Animal Use Ethics Committee, No. 0136/2017). For the PCR assay, we used Illustra Bacteria Mini Spin Kit for bacterial DNA. At this moment, we have already tested sea-see, seg-ser, sew, and sex, and the results have already been submitted to Fisher Exact Probability Test or Chi-square Test. Considering the isolates obtained from clinical mastitis, the most frequent enterotoxins were selw (99%), selx (78%) and selh (50.5%), and sec, see, sej, sell, selp,and ser were absent. Among the subclinics, selw (82.5%) selm (15.5%) and selx (14.6%) were the most frequent, and sea-see, seg, sei-sel, sem-ser were absent. We have already observed statistically significant differences for seb, seg, seh, sei, selo, selu, selw and selx. Other interesting results were the low number of genes in each isolate from subclinical mastitis [0 genes: 14 (13.6%); 1 gene: 55 (53.4%); 2 genes: 33 (32%) or 3: 1 (0.97%)] compared to clinical isolates [1 gene: 5 (4.9%); 2 genes: 29 (28.1%); 3 genes: 38 (36.9%); 4 genes: 14 (13.6%); 5 genes: 5 (4.9%); 6 genes: 4 (3.9%); 7 genes: 5 (4.9%); 8 genes: 2 (1.9%) and 9 genes: 1 (1%)]. Based on these results, we can conclude that enterotoxins indeed play an important role in clinical signs in cattle with mastitis.

Keywords: mastitis, S. aureus, PCR, staphylococcal enterotoxin

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Authors: Martins A. Adefisoye, Mpaka Lindelwa, Fadare Folake, Anthony I. Okoh

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Evolution and rapid dissemination of antibiotic resistance from one ecosystem to another has been responsible for wide-scale epidemic and endemic spreads of multi-drug resistance pathogens. This study assessed the prevalence of Enterobacteriaceae in different environmental samples, including river water, hospital effluents, abattoir wastewater, animal rectal swabs and faecal droppings, soil, and vegetables, using standard microbiological procedure. The identity of the isolates were confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrophotometry (MALDI-TOF) while the isolates were profiled for resistance against a panel of 16 antibiotics using disc diffusion (DD) test, and the occurrence of resistance genes (ARG) was determined by polymerase chain reactions (PCR). Enterobacteriaceae counts in the samples range as follows: river water 4.0 × 101 – 2.0 × 104 cfu/100 ml, hospital effluents 1.5 × 103 – 3.0 × 107 cfu/100 ml, municipal wastewater 2.3 × 103 – 9.2 × 104 cfu/100 ml, faecal droppings 3.0 × 105 – 9.5 × 106 cfu/g, animal rectal swabs 3.0 × 102 – 2.9 × 107 cfu/ml, soil 0 – 1.2 × 105 cfu/g and vegetables 0 – 2.2 × 107 cfu/g. Of the 700 randomly selected presumptive isolates subjected to MALDI-TOF analysis, 129 (18.4%), 68 (9.7%), 67 (9.5%), 41 (5.9%) were E. coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp. respectively while the remaining isolates belong to other genera not targeted in the study. The DD test shows resistance ranging between 91.6% (175/191) for cefuroxime and (15.2%, 29/191) for imipenem The predominant multiple antibiotic resistance phenotypes (MARP), (GM-AUG-AP-CTX-CXM-CIP-NOR-NI-C-NA-TS-T-DXT) occurred in 9 Klebsiella isolates. The multiple antibiotic resistance indices (MARI) the isolates (range 0.17–1.0) generally showed >95% had MARI above the 0.2 thresholds, suggesting that most of the isolates originate from high-risk environments with high antibiotic use and high selective pressure for the emergence of resistance. The associated ARG in the isolates include: bla TEM 61.9 (65), bla SHV 1.9 (2), bla OXA 8.6 (9), CTX-M-2 8.6 (9), CTX-M-9 6.7 (7), sul 2 26.7 (28), tet A 16.2 (17), tet M 17.1 (18), aadA 59.1 (62), strA 34.3 (36), aac(3)A 19.1 (20), (aa2)A 7.6 (8), and aph(3)-1A 10.5 (11). The results underscore the need for preventative measures to curb the proliferation of antibiotic-resistant bacteria including Enterobacteriaceae to protect public health.

Keywords: enterobacteriaceae, antibiotic-resistance, MALDI-TOF, resistance genes, MARP, MARI, public health

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Authors: Maneesh Kumar, Roshan Kamal Topno, Manas Ranjan Dikhit, Vahab Ali, Ganesh Chandra Sahoo, Bhawana, Major Madhukar, Rishikesh Kumar, Krishna Pandey, Pradeep Das

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Chandipura vesiculovirus is an emerging (-) ssRNA viral entity belonging to the genus Vesiculovirus of the family Rhabdoviridae, associated with fatal encephalitis in tropical regions. The multi-functionally active viral RNA-dependent RNA polymerase (vRdRp) that has been incorporated with conserved amino acid residues in the pathogens, assigned to synthesize distinct viral polypeptides. The lack of proofreading ability of the vRdRp produces many mutated variants. Here, we have performed the evolutionary analysis of 20 viral protein sequences of vRdRp of different strains of Chandipura vesiculovirus along with other viral species from genus Vesiculovirus inferred in MEGA6.06, employing the Neighbour-Joining method. The p-distance algorithmic method has been used to calculate the optimum tree which showed the sum of branch length of about 1.436. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1000 replicates), is shown next to the branches. No mutation was observed in the Indian strains of Chandipura vesiculovirus. In vRdRp, 1230(His) and 1231(Arg) are actively participated in catalysis and, are found conserved in different strains of Chandipura vesiculovirus. Both amino acid residues were also conserved in the other viral species from genus Vesiculovirus. Many isolates exhibited maximum number of mutations in catalytic regions in strains of Chandipura vesiculovirus at position 26(Ser→Ala), 47 (Ser→Ala), 90(Ser→Tyr), 172(Gly→Ile, Val), 172(Ser→Tyr), 387(Asn→Ser), 1301(Thr→Ala), 1330(Ala→Glu), 2015(Phe→Ser) and 2065(Thr→Val) which make them variants under different tropical conditions from where they evolved. The result clarifies the actual concept of RNA evolution using vRdRp to develop as an evolutionary marker. Although, a limited number of vRdRp protein sequence similarities for Chandipura vesiculovirus and other species. This might endow with possibilities to identify the virulence level during viral multiplication in a host.

Keywords: Chandipura, (-) ssRNA, viral RNA-dependent RNA polymerase, neighbour-joining method, p-distance algorithmic, evolutionary marker

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Authors: Andrea Parisi, Samantha Vilkins, Luis Furuya-Kanamori, John A. Crump, Benjamin P. Howden, Darren Gray, Kathryn Glass, Martyn Kirk

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Objectives: Salmonella is a leading cause of foodborne enterocolitis worldwide. Nontyphoidal Salmonella (NTS) infections that are Multi-Drug Resistant (MDR) (non-susceptible to ≥1 agent in ≥3 antimicrobial categories) may result in more severe outcomes, although these effects have not been systematically examined. We conducted a systematic review and meta-analysis to examine impacts of MDR NTS on health in high-income settings. Methods: We systematically reviewed the literature from scientific databases, including PubMed, Scopus and grey literature sources, using PRISMA guidelines. We searched for data from case-control studies, cohorts, outbreaks, reports and theses, imposing no language restriction. We included only publications from January 1990 to September 2016 from high income countries as classified by World Bank. We extracted data from papers on duration of illness, hospitalisation rates, morbidity and mortality for MDR and non-MDR NTS strains. Results: After removing duplicates, the initial search revealed 4258 articles. After further screening, we identified 16 eligible studies for the systematic review, and 9 of these were included in meta-analysis. NTS serotypes differed among the reported studies but serotype Typhimurium, Enteritidis, Newport and Heidelberg were among the most often reported as MDR pathogens. Salmonella infections that were MDR were associated with excess bloodstream infections (OR 1.63; 95%CI 1.18-2.26), excess hospitalisations (OR 2.77; 95%CI 1.47-5.21) and higher mortality (OR 3.54; 95%CI 1.10-11.40). Conclusions: MDR NTS infections are a serious public health concern. With the emergence of MDR Salmonella strains in the high-income countries, it is crucial to restrict the use of antimicrobials both in animals and humans, and intervene to prevent foodborne infections.

Keywords: Antimicrobial Resistance, Bloodstream Infection, Health Outcomes, Hospitalisation, Invasive Disease, Multi-Drug Resistance (MDR), Mortality, Nontyphoidal Salmonella

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Authors: Albert P. Ekanem, Austin I. Obiekezie, Elizabeth X. Ntia

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Introduction: Some African plants and bile from animals have shown efficacies in the treatment and control of diseases in farmed fish. The background of the study is based on the fact the African rain forest is blessed with the abundance of medicinal plants that should be investigated for their use in the treatment of diseases. The significance of the study is informed by the fact that chemical-based substances accumulate in the tissues of food fish, thereby reducing the food values of such products and moreover, the continuous use of chemotherapeutics in the aquatic environments tends to degrade the affected environment. Methodology: Plants and animal products were extracted, purified and applied under in vitro and in vivo conditions to the affected organisms. Effective plants and bills were analyzed for biologically active substances responsible for the activities by both qualitative and HPLC methods. Results: Extracts of Carica papaya and Mucuna pruriens were effective in the treatment of Ichthyophthiriasis in goldfish (Carassius auratus auratus) with high host tolerance. Similarly, ectoparasitic monogeneans were effectively dislodged from the gills and skin of goldfish by the application of extracts of Piper guineense at therapeutic concentrations. Artemesia annua with known antimalarial activities in human was also effective against fish monogenean parasites of Clarias gariepinus in a concentration-related manner without detriments to the host. Effective antibacterial activities against Aeromonas and Pseudomonas diseases of the African catfish (Heterobranchus longifilis) were demonstrated in some plants such as Phylanthus amarus, Allium sativum, A. annua, and Citrus lemon. Bile from some animals (fish, goat, chicken, cow, and pig) showed great antibacterial activities against some gastrointestinal bacterial pathogens of fish. Conclusions: African plants and some animal bile have shown potential promise in the treatment of diseases in fish and other aquatic animals. The use of chemical-based substances for control of diseases in the aquatic environments should be restricted.

Keywords: control, diseases, fish, treatment

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Authors: C. Miranda, R. Soares, S. Cunha, L. Ferreira, G. Igrejas, P. Poeta

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Different animal species, including ornamental animals, are reported as potential reservoirs of antibiotic resistance genes. Consequently, these resistances can be disseminated in the environment and transferred to humans. Moreover, multidrug-resistant bacteria reduce the efficacy of antibiotics, as the case of vancomycin-resistant enterococci. Enterococcus faecalis and E. faecium are described as the main nosocomial pathogens. In this line, the aim of this study was to characterize resistance and virulence genes of enterococci species isolated from samples of food supplied to ornamental animals during 2020. The 29 enterococci isolates (10 E. faecalis and 19 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB and ermC), tetracycline (tetL, tetM, tetK and tetO), quinupristin/dalfopristin (vatD and vatE), gentamicin (aac(6’)-aph(2’’)-Ia), chloramphenicol (catA), streptomycin (ant(6)-Ia) and vancomycin (vanA and vanB). The same isolates were also tested for 10 virulence factors genes (esp, ace, gelE, agg, fsr, cpd, cylA, cylB, cylM and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. The most prevalent resistance genes detected in both enterococci species were ermB (n=15, 52%), ermC (n=7, 24%), tetK (n=8, 28%) and vatE (n=4, 14%). Resistance genes for vancomycin were found in ten (34%) E. faecalis and ten (34%) E. faecium isolates. Only E. faecium isolates showed the presence of ermA (n=2, 7%), tetL (n=13, 45%) and ant(6)-Ia gene (n=4, 14%). A total of nine (31%) enterococci isolates were classified as multidrug-resistant bacteria (3 E. faecalis and 6 E. faecium). In three E. faecalis and one E. faecium were not detected resistance genes. The virulence genes detected in both species were agg (n=6, 21%) and cylLL (n=11, 38%). In general, each isolate showed only one of these virulence genes. Five E. faecalis and eleven E. faecium isolates were negative for all analyzed virulence genes. These preliminary results showed the presence of multidrug-resistant enterococci in food supplied to ornamental animals, in particular vancomycin-resistant enterococci. This genotypic characterization reinforces the relevance to public health in the control of antibiotic-resistant bacteria.

Keywords: antibiotic resistance, enterococci, feed, ornamental animals

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