Abstract Rapid detection of virulent pathogens during an outbreak is critical for public health advisories and control of the disease in a population. While many mo lecular techniques for point of care and clinical diagnosis abound, the US ex perience with the COVID-19 testing in the early stages of the pandemic un derscores the critical importance of determining the appropriate target gene(s) with in-built controls that reliably detect pathogens with high sensitivity and specificity. Assays and research for diagnostics and therapy could be slowed during an epidemic because access to the required BSL-3 and BSL-4 laborato ries are limited. So, during the 2014 West Africa Ebola outbreak, we tested the hypothesis that using synthetic cDNA of Ebolavirus in a bacteria surro gate (fit for all lab settings), would remain unmutated and safe after several generations, serving as an effective positive control in research settings, self test and point-of-care detection platforms. Primers were designed for the de tection and quantification of the nucleoprotein (NP) gene of the 2014 Mako na Ebola strain (KR781608.1, 733 - 1332 bp). To test the stability of artificially inserted translation arrest in the Orf of the model gene, it was edited to in clude three STOP codons in the RNA transcript using SNAP GENE. The segment was then spliced into a high copy number plasmid, cloned into One ShotTM TOP10 Escherichia coli (Invitrogen), and tested for stability and safety by periodic subculture, extraction and sequencing. Unlike COVID-19, rapid detection of blood-borne etiologies like Ebola requires optimized protocols for blood matrix. Using real-time PCR and newly designed primer pairs, the EBOV surrogate was detected and enumerated in human blood and regular broth and buffers. Based on aligned sequence analysis, the EBOV synthetic NP gene was stable (>99.9999% similarity coefficient) for at least 3 months. Detection sensitivity in broth and blood was at least 100 cells/ml or about 5.8 × 103 to 7.3 × 103 virion equivalents per ml. While the developments of tran scription-and-replication-competent virus like particles (trVLP) have made it possible to study the infection and replication cycles of virulent pathogens in BSL-2 laboratories, the simplicity of our model and the reproducibility of de tection and enumeration show the utility of synthetic bio-components as pos itive controls for point of care diagnostic tools. The inserted stop codons re mained intact after many generations, suggesting that expressed virulent pro teins can be easily silenced in synthetic biology models for research in BSL-1 and 2 and a wide range of pathogens. Synthetic bio-components can thereby aid further research by reducing costs and improving safety for workers and stakeholders.

Abstract "Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of cap sular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 sam ples were collected from clinically sick (n = 107) and pneumonic lung tis sue (n = 59). Laboratory assay confirmed isolation of M. haemolytica 37 (22.29%), P. multocida 25 (15.06%), B. trehalosi 12 (7.23%), and H. somni 15 (9.04%). PCR assay of P. multocida capsular typing revealed 21 (84.0%) cap A (hyaD-hyaC) and 4 (16.0%) cap D (dcbF) strains. M. haemolytica se rotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. P. multocida biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, re spectively. M. haemolytica harbored more than 60% ssa gene, and 90.91% sodA while FbpA, TbpA, and lktC genes were found in all isolates. Likewise, all P. multocida exhibited toxA, FbpA, TbpA, and pmSLP genes. The current finding showed that M. haemolytica serotype A: 1 is frequently associated with BRD followed by P. multocida biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, viru lence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak sur veillance and molecular approaches are indispensable in designing efficient prevention strategies."

Abstract In 2019, the coronavirus pandemic broke out as a serious public health issue worldwide. In Côte d’Ivoire, the number of cases of COVID-19 has increased rapidly. The Severe Acute Respiratory Syndrome virus (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) receptors in the respiratory tracts and enters the respiratory and alveolar cells of infected patients. Deficiency of fat-soluble vitamin D3 is associated with respiratory distress syndrome and pulmonary fibrosis by activation of the renin-angiotensin system. In Côte d’Ivoire, very little research is being done on SARS-CoV-2 and vitamin D. The objective of this study was to assess the vitamin D status of people infected and suffering from COVID-19 in order to contribute to their medical treatment. The study involved 100 adults infected with SARS-CoV-2 (24 women and 76 men). After confirmation of the patient’s SARS-CoV-2 status by RT-PCR, the 25 (OH) vitamin D assay was performed on the Cobas 6000 device and compared to control subjects, the non-COVID-19 positive. A significant decrease in 25-hydroxy vitamin D3 concentrations (44 ± 1.29 nmole/L)was observed in patients infected with SARS-CoV-2, compared to control (78 ± 0.68 nmole/L) (p < 0.0001). The 25-hydroxy vitamin D3 deficiency requires vitamin D supplementation in the management of hospitalized patients infected with SARS-CoV-2.

Abstract Poultry chickens are potential source of transmission of zoonotic Salmonella, into human food chain, causing food-borne illness and also hindering development of poultry industry in Bangladesh. The non-judicious uses of antibiotics in poultry farm have increased the multidrug resistant bacteria. So, this study reports the occurrence of Salmonella in poultry samples (meat, egg, liver and cloacal swab) and the antimicrobial resistance pattern of the isolates. This study was carried out throughout the period of May 2019-March 2020,at the bacteriological laboratory in the Department of Microbiology, University of Chittagong. Isolates were identified on the basis of cultural and biochemical tests from a total of 25 broiler samples (meat, liver, eggshell and cloacal swab). Antibiotic susceptibility pattern was observed using Kirby-Bauer disk diffusion method. The overall detection rate of Salmonella was 48% (12/25) and the highest occurrence was noticed in raw meat 62.5% and the lowest in liver (37.5%). The antimicrobial resistance tests revealed that all the isolates (n = 12) exhibited 100% resistance to vancomycin and cephalexin, followed by ampicillin (75%), nalidixic acid (58.33%), chloramphenicol (41.66%), doxycycline (50%), and neomycin (50%). On the other hand, ciprofloxacin showed 83.33%, ceftazidime and amoxicillin showed 91.6% sensitivity respectively. A considerably high proportion of isolates (11/12, 91.67%) was resistant to three or more antibiotics and 6 multidrug profiles were observed. The ampicillin-chloramphenicol-nalidixic acid-neomycin-cephalexindoxycycline-vancomycin (4/12) was more frequently observed phenotype in multidrug profiles. Finally, two multidrug-resistant strains of Salmonella were identified and classified based on their 16S ribosomal RNA gene sequences as Salmonella enterica subsp. enterica strain Eshaa2 and Salmonella enterica subsp. enterica strain Eshiika3 at NCBI GenBank with Accession no. MT163513 and MT164531 respectively. So, more attention should be focused on increasing antibiotic surveillance to cope with the spread of emerging resistance and on the alternative approaches.

Abstract Background: Metagenomics approaches are increasingly being utilized as “dipstick” for microbial carriage. In this study, 16S rRNA metagenomics was used to probe for microbial community that resides in the ticks, those they pick from the environment, wildlife and livestock and to identify potential tick borne zoonoses. Methods: Tick DNA from 463 tick pools collected from domestic animals between 2007 and 2008 were amplified with primers that target the 16S rRNA V3-V4 domain and then sequenced on Illumina Miseq platform using 300 cycles version 3 kits. Ticks were pooled according to species and animal from which they were collected. A non-target control was used to track laboratory contaminants. Sequence data were analyzed using Mothur v1.3 pipeline and R v3.3.1 software and taxonomy determined using SILVA rRNA database. Shannon diversity index was used to compute bacterial diversity in each tick species before computing the means. Results: A total of 645 bacteria genera grouped into 27 phyla were identified. Four phyla contributed 97.4% of the 36,973,934 total sequences. Proteobacteria contributed 61.2% of these sequences that tarried to 33.8% genera, compared to 15.9% (23.4% genera) for Firmicutes, 15.6% (20% genera) for Actinobacteria and 4.7% (11.6% genera) for Bacteroidetes. The remaining 23 phyla only contributed 2.6% of the sequence reads (11.2% genera). Amongst the 645 genera, three groups were discernible, with the biggest group comprised commensals/symbionts that contributed 93.6% of the genera, but their individual sequence contribution was very low. Group two comprised genera that are known to contain pathogenic species, with Coxiella contributing 15,445,204 (41.8%) sequences, Corynebacterium (13.6%), Acinetobacter (4.3%), Staphylococcus (3.9%), Bacillus (2.7%) and Porphyromonas (1.6%), Ralstonia (1.5%), Streptococcus (1.3%), Moraxella (1.3%), amongst others. Group three comprised genera known to contain tick borne zoonotic pathogens (TBZ): Rickettsiae, Anaplasma, Francisella, Ehrlichia, Bartonella and Borrelia. Individually the TBZ contributed <1% of the sequences. By Shannon diversity index, Amblyomma variegatum carried the least diverse bacteria (mean Shannon diversity index of 2.69 ± 0.92) compared to 3.79 ± 1.10 for A. gemma, 3.71 ± 1.32 for A. hebraeum, 4.15 ± 1.08 for other Amblyomma spp, 3.79 ± 1.37 for Hyalomma truncatum, 3.67 ± 1.38 for other Hyalomma spp, 3.86 ±1.27 for Rhipicephalus annulatus, 3.56 ± 1.21 for Rh. appendiculatus, and 3.65 ± 1.30 for Rh. Pulchellus, but the difference was not significant (p = 0.443). Conclusion: This study illustrates the utility of 16S rRNA metagenomics in revealing the complexity of bacteria communities that reside and/or transit through the tick having been picked from the environment, livestock and/or wild animals, some with potential to cause zoonoses.

Abstract Chromobacterium violaceum is a Gram negative, facultative anaerobe, generally present in water, soil in tropical and subtropical regions. This bacterium is an emerging environmental pathogen that causes life threatening infection in humans and animals. It can cause wound infection, visceral abscess, septicaemia, meningitis, diarrhoea, UTI. It is associated with significant mortality due to severe systemic infection. As the bacteria have high spreading tendency leading to sepsis, early identification and prompt treatment is necessary. Here we report a case of Chromobacterium violaceum wound infection in a 9 years old male from Dhaka, who was successfully treated with combination of cefixime and flucloxacillin antibiotics as per culture sensitivity report.

Abstract Introduction: Chronic kidney disease (CKD) is a major cause of death in sub-Saharan Africa. The effects of the CKD on the host and the continuous therapeutic measures increase the hypothesis of blood-borne diseases transmission. Objective: This study aimed to find the frequency of occult hepatitis B virus (OBI) in patients of chronic renal failure (CRF) and to study the possibilities of infection acquisition. Methods: During 2017 and 2019, two hundred CRF patients under regular haemodialysis and attending Gezira Hospital for Renal Diseases and Surgery were recruited. Plasma specimens were collected and used for detection of hepatitis B surface antigens (HbsAg), total hepatitis B core antibodies (anti-HBc) and hepatitis B virus DNA isolation. Nested PCR reaction was followed to identify HBV. Socio-clinical data for each participant was obtained. Results: Male patients represented 64% (128/200), most frequent age group was from 41 to 60 years with percentage of 56.5% (113/200), 86% (172/200) of CRF patients were received blood while 42% (84/200) get HBV vaccination. Hepatitis B core antibodies were found in 54% (108/200) of studied cases, and 22% (42/188) of tested DNA were positively amplified for target gene. Detection of Hepatitis B core antibodies was significantly associated with marital status while absence of vaccination significantly associated with the detection of both hepatitis B core antibodies and HBV DNA. Conclusion: This study found high frequency of OBI in CRF patients, to reduce the transmission of the disease, possible hypotheses should be studied, including blood transfusion, haemodialysis process and HBV vaccination status.

Abstract The present opinion article, while dealing with the debated origin of “Severe Acute Respiratory Syndrome-Coronavirus-2” (SARS-CoV-2), the betacoronavirus responsible for “Corona Virus Disease-2019” (COVID-19), provides a speculative insight into the so-called “gain of function” (GOF), a process resulting in the acquirement of new phenotypic features on behalf of the viral pathogen. More in detail, a GOF-related phenomenon leading to increased SARS-CoV-2 virulence and/or transmissibility—as clearly exemplified by the “delta” and the “omicron” as well as by other “variants of concern”—would not necessarily imply that viral genetic manipulations made in the laboratory are its exclusive drivers, provided that GOF may also occur as a consequence of a natural selection process. In order to gain a better understanding of SARS-CoV-2 GOF and GOF-associated phenomena, an in-depth knowledge of the complex viral-host interaction dynamics is absolutely needed, while also paying special attention to the human-animal-viral ecological interfaces within an “ad hoc” multidisciplinary, “holistic”, scientific evidence-based and “One Health”-based approach.

Abstract Aspergillus species and aflatoxins production are more prevalent during times of high heat and drought. In South Africa, there is frequent occurrence of drought as a result of climate change. The aim of this study was to investigate the biodiversity and distribution of Aspergillus species with their corresponding toxins in maize from main maize producing regions of South Africa;[Western Regions (WR) and Eastern Regions (ER)]. One hundred and twenty-three (64 from WR and 59 from ER) maize samples from the two agroclimatic regions in South Africa were analyzed using cultural, molecular and analytical methods. Across agro-climatic regions, Aspergillus species contaminated about 62% of the maize samples, while Aspergillus flavus was the most prevalent (47.15%) followed by Aspergillus fumigatus (4.69%) while the least was Aspergillus parasiticus (0.81%). The Western Regions showed a higher distribution of varieties of Aspergillus species compared to the Eastern Regions. Aflatoxins contaminated only 27.64% of the maize samples with a mean total aflatoxin concentration of 2.40 μg/kg which is below the South Africa’s set standard for total aflatoxin in food (5 μg/kg). About 10.57% of the samples produce aflatoxins above the 5 μg/kg permissible limit for total aflatoxin in foods. The ratio of toxigenic to atoxigenic strains of Aspergillus flavus was generally low in all the regions of South Africa. This study could aid policy makers to make informed decisions in developing remediation strategies for Aspergillus mycotoxins.

Abstract An investigation was made to survey the possible presence of Staphylococcus aureus, Escherichia coli and Salmonella spp. from fast-food shops in Al-Ahsa Province, Kingdom of Saudi Arabia (KSA), as potential reservoir of human infection and antimicrobial resistance. A total of 100 samples of shawarma poultry meat were collected from different localities of the province. Conventional, commercial VITEK 2 and molecular techniques were used for isolates’ identification and antibiogram detection. Staph aureus was isolated at a rate of 14% and CNS as Staph. sciuri and Staph. xylosus at 2%. E. coli was identified at a rate of 12% and antibiogram analysis showed 41.67% of isolates to be extended-spectrum β-lactamases (ESBL) with evidence of multi-drug resistance (MDR). Molecular analysis of E. coli revealed presence of sero-groups O1 and O2, entero-toxigenic (ETEC), shiga-toxigenic, ST540 and the prototypical ETEC strain H10407 which are potential public health hazard. Salmonella enterica serovar Enteritidis showed 19% prevalence while S. Typhimurium with 8% prevalence. Anti-microbial sensitivity of 15 strains of S. Enteritidis and 5 strains of S. Typhimurium showed multi-drug resistance (MDR).

Abstract Vinegar production is seriously affected by the sensitivity of acetic acid bacteria (AAB) to high temperature, high ethanol concentrations, and high acetic acid concentrations. The aim of this study was to investigate the thermo-ethanol-acid tolerance characters of five AAB strains (VMA1, VMA5, VMA7, VMAM, VMAO) previously isolated from fermented mango alcohol and belonging to Gluconoacetobacter genera. As result, the five AAB strains exhibited good growth and acid production at temperatures up to 45˚C; they could tolerate and produce acetic acid at ethanol concentrations up to 20% (v/v). In addition, the studied strains showed growth at acetic acid concentrations up to 4.5% (w/v). Strains VMA7 and VMAO showed the highest resistance properties: they demonstrated acid production at 50˚C and VMAO could even grow at 60˚C; they tolerated and produced acetic acid at 25% (v/v) ethanol concentration; they showed resistance to acetic acid concentrations up to 6% (w/v). Considering all these properties, the use of these strains would seriously contribute to improving the quality of the vinegar produced and help to reduce the cooling water feeds in vinegar production especially in hot countries in the context of global warming.

Abstract Sporotrichosis is a subcutaneous mycosis caused by the Sporothrix schenckii complex. It has three classic clinical variants: fixed, lymphangitic, and systemic. Treatment in most cases has been itraconazole or potassium iodide. The aim of this paper is to communicate an unusual relapsing case treated with IK. We report a 73-year-old woman with lymphangitic sporotrichosis, adequate response to treatment with potassium iodide, and recurrence 15 months after. A molecular test was performed through the amplification of a 331 bp fragment of the calmodulin gene. In both infections, the same specie was isolated. The effects of potassium iodide are briefly discussed, and we conclude that the same treatment can be prescribed, if no side effects are observed.

Abstract Vegetable oil spills are becoming frequent and are potentially more challenging than petroleum hydrocarbon spills. Microbial lipases occupy a place of prominence among biocatalysts and are often used for the remediation of vegetable oil spills. There is a need for extensive characterisation of lipase for the treatment of vegetable oil-polluted sites. This work was carried out to monitor the degradation pattern of vegetable oil. Two microbial isolates previously isolated from an oil mill in Ibadan, Nigeria were used for the bioremediation experiment. Soil samples (with some purposely contaminated with 2 different vegetable oils) collected from the Nursery section of the Microbiology department as well as soil samples from the oil mill were all subjected to varied treatment processes. Field bioremediation using the isolates was carried out for 12 weeks. The isolates were identified, and microbial load and residual oil weight were determined during the degradation period using standard methods. The two isolates were identified as Pseudomonas fluoresecens and Candida parapsilosis. The result of sterile soil samples with and without mixing option, from palm oil and palm kernel purposely contaminated soils for all the various treatments, showed a general increase in total viable counts from the 2nd week to the 12th week, however in the non-sterile counterpart there was a steady increase from the 2nd week to the 8th week and subsequently, a gradual decrease from the 10th to the 12th week. The residual oil weight in the sterile purposely palm oil-contaminated soil, treated with the consortium (POC) non-mixed gave a reduction of value from 0.335 g on day 0 to 0.13025 g by the 12th week. From the oil mill non-sterile, treated with (POC) non-mixed sample, the residual weight after 12 weeks of treatment was 0.0043 g from an initial weight of 0.01 g. The microorganisms Pseudomonas fluoresecens and Candida parapsilosis had the potential for the degradation of fatty waste. They could therefore be employed in the environmental cleanup of the vegetable oil spill sites.

Abstract Noroviruses are positive-sense, single-stranded, non-enveloped RNA virus that measures approximately 27 - 35 nm in diameter. It affects humans of all ages and races causing most cases of viral gastroenteritis worldwide. Infection results from ingestion of contaminated food or water as well as causing diarrhea and vomiting in humans. Extracts from plants are known to have antioxidant, anti-inflammatory, and adhesive properties which are associated with barrier functions. The aim of this study was to elucidate whether plaque reduction was due to an effect of methanolic plant extract directly on the virus, whether the extract affects viral replication, and lastly, whether the extract disrupts the cell surface binding with the virus. The plant extracts of interest were the calyces of Hibiscus sabdariffa (HS) and the seeds of Zanthoxylum armatum (ZA). Antiviral activities of these extracts were determined against murine norovirus. The logarithmic viral reduction per plaque-forming unit was (22 log10) PFU/ml (control), (15 log10) PFU/ml (treated HS), and (12 log10) PFU/ml (treated ZA) with a significant reduction (68% and 55% respectively) when compared with the control for the direct effect on the virus. The role of extracts on virus replication showed (25 log10) PFU/ml (control) as against the HS treated-virus-infected cells (9 log10) PFU/ml and ZA treated-virus-infected cells (5 log10) PFU/ml (36% and 20% respectively). Finally, effect of the extract on the viral attachment showed (31 log10) PFU/ml (control), (12 log10) (HS-treated) and (9 log10) PFU/ml (ZA-treated), (39% and 29% respectively. Extract treatment with HS and ZA has shown evidence of a reduced number of plaques formation with the latter having fewer plaques. Both extracts have proven potential to reduce the viral multiplication process by interfering with the replication process. This study shows that Hibiscus sabdariffa (calyces) and Zanthoxylum armatum (seed) extracts disrupt murine norovirus from consistent viral replication.

Abstract Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).