Molecular diagnosis of hen eggs microbiota.

The study was conducted in the Kingdom of Saudi Arabia from 2020 and 2022. The identification, characterization, and evaluation of microbes found in hen eggs was done and it was found very important to prevent contamination caused by various harmful pathogenic microbes. It was found that contaminated eggs harbor various harmful microbes which affect health due to multiple infectious diseases. Hen eggs contain a wide variety of microbes, and several distinct approaches were utilized as well as available for achieving detailed pathogenic information. The information obtained is highly essential for people who consume eggs as a food product.  It is of the utmost importance to protect people from getting sick due to the consumption of contaminated eggs or eggs from chickens that have been infected by various harmful pathogens.  During the experiment, we found that eggs were contaminated directly or the chicken that laid the egg was contaminated. Using molecular genetic analysis, it is possible to detect pathogenic and non-pathogenic contaminations in eggs.  During present studies, the cutting-edge molecular techniques of 16S rRNA gene sequencing technology were used to carry out the objective of performing a molecular identification of the microbial communities infecting eggs. The present research is aimed at determining whether the microbial communities in hen eggs are harmful to humans. The results further indicated most bacteria have the potential to cause illness in humans including Escherichia fergusonii, Salmonella enterica, Pseudocitrobacter faecalis, Yakenella regensburgei, and Erwinia pyrifoliae. Further, research suggested that eggs need to be properly cooked and thoroughly washed to eliminate the possibility of consuming infected eggs.

Molecular diagnosis for camel raw milk microbiota.

The existence of diverse microbes in unprocessed camel milk poses a significant threat to the well-being of a large population, especially infants and toddlers. The objective of this study was to ascertain the existence of microorganisms in unprocessed raw camel milk by employing a molecular-based technique in combination with a histological examination of bacteria. The identification of microbial species was achieved by employing PCR amplification and sequencing of 16s rRNA gene fragments. Various micorganisms found includes the probiotic Lactobacillus species, Staphylococcus succinic, Macrococcus casealyticus, Bacillus cohnii, and Salinicoccus kunmingensis. To prevent microbial contamination in raw milk, it is necessary to adequately heat or pasteurise the milk and to wash and sterilise the udder before milking the camel. This is because raw milk contains microbes that cause multiple diseases. Moreover, in the current era of the COVID-19 pandemics, ensuring proper sanitary conditions in milk and its derivatives might potentially mitigate the transmission of various diseases among consumers shortly. Keywords: camel, microbiota, 16s rRNA gene, PCR.

Comparative molecular studies of halophilic bacteria from saline water and soil in the Saudi environment / Estudos moleculares comparativos de bactérias halofílicas de água salina e solo no meio ambiente saudita

Halophilic bacteria are microorganisms that grow optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents. Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates were screened by plate assay on starch and gelatin agar plates for amylase and protease, respectively. Two bacterial isolates i.e. Bacillus glycinifermentans S3 and Enterobacter cloacae W1were found to possess significant amylase and protease activity.

Bactérias halofílicas são microrganismos que crescem de maneira ideal na presença de uma concentração muito alta de cloreto de sódio. Halófilos são fontes vitais de várias enzimas, incluindo hidrolases, que são muito estáveis e cataliticamente altamente eficientes em alta concentração de sal e outras condições extremas, como alta temperatura, pH e presença de solventes orgânicos. Várias hidrolases como amilases, proteases e lipases foram obtidas a partir de bactérias halofílicas e são comumente usadas para várias aplicações industriais. Iniciamos uma triagem para isolar e caracterizar as bactérias halofílicas do Mar Vermelho, que é um dos corpos de água mais salgados do mundo. Amostras de água e solo, coletadas na costa do Mar Vermelho, Jeddah, na Arábia Saudita, foram examinadas quanto ao isolamento de bactérias halofílicas. Foram obtidos dez isolados bacterianos, caracterizados por testes bioquímicos e seqüenciamento do gene 16S rRNA. As bactérias produtoras de hidrolase entre os isolados foram triadas por ensaio em placa em placas de amido e ágar de gelatina para amilase e protease, respectivamente. Verificou-se que dois isolados bacterianos, isto é, Bacillus glycinifermentans S3 e Enterobacter cloacae W1, possuíam significativa atividade de amilase e protease.

Risk factors and molecular features of extended-spectrum beta-lactamase producing bacteria at southwest of Saudi Arabia.

OBJECTIVES: To explore the risk factors, the prevalence rate, and gene types of extended-spectrum beta-lactamase (ESBL)-producing bacteria as the causative agents of infection at King Abdulaziz Specialist Hospital (KAASH), Taif, Kingdom of Saudi Arabia. Methods: This was a retrospective study conducted during the period between February 2017 and January 2018. All samples obtained from the KAASH were analyzed. The MicroScan Walkaway System, bacteriological examination and double disk synergy tests were used to detect ESBL-producing bacteria. To identify ESBL genes, the polymerase chain reaction (PCR) technique was used. Results: The ESBL phenotype was detected in 351 of 1151 isolates (30.5%); Escherichia coli (E. coli) (62.7%) and Klebsiella pneumoniae (K. pneumoniae) (23.6%) were the most prevalent. The highest proportion of ESBL specimens was found in urine (62%.5), and these organisms were mainly isolated from the female medical ward (20.2%). Based on the statistical analysis, lung diseases, renal diseases, diabetes and heart diseases contributed to the spread of ESBL infections. Amikacin, imipenem, meropenem and tigecycline were found to be effective in overcoming ESBL infections; however, these antibiotics may be inappropriate for new strains of K.pneumoniae. The distribution of the blaCTX-M gene was high (87%), compared with blaTEM (74.9%) and blaSHV (29.4%).   Conclusion: These data provide new epidemiological information about the prevalence of ESBL-producing organisms among patients in KAASH, Taif, Saudi Arabia. In addition, this study identified the clonal nature of isolated E.coli and K.pneumoniae.

Transcription and regulation of hepatitis B virus genes in host sperm cells.

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo.

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Cell factories for insulin production.

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

A sensitive and rapid assay for investigating vertical transmission of hepatitis B virus via male germ line using EGFP Vector as reporter.

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed.

An improved experimental model for studying vertical transmission of hepatitis B virus via human spermatozoa.

Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.