ARTICLE | doi:10.20944/preprints202005.0153.v2
Subject: Life Sciences, Microbiology Keywords: Escherichia coli; antimicrobial resistance; ESBL; MDR; frozen chicken meat; Bangladesh
Online: 27 May 2020 (08:31:20 CEST)
Escherichia coli is known as one of the most important foodborne pathogens in humans, and contaminated chicken meat is an important source of foodborne infection with this bacterium. The occurrence of extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-Ec), in particular, in chicken meat is considered a global health problem. This study aimed to determine the magnitude of E. coli, with special emphasis on ESBL-Ec, along with their phenotypic antimicrobial resistance pattern in frozen chicken meat. The study also focused on the determination of ESBL-encoding genes in E. coli. A total of 113 frozen chicken meat samples were purchased from 40 outlets of nine branded supershops in five megacities in Bangladesh. Isolation and identification of E. coli were done based on cultural and biochemical properties, as well as PCR assay. The resistance pattern was determined by the disc diffusion method. ESBL-encoding genes were determined by multiplex PCR. The results showed that 76.1% of samples were positive for E. coli, of which 86% were ESBL producers. All the isolates were multidrug-resistant (MDR). Resistance to 9–11 and 12–13 antimicrobial classes was observed in 38.4% and 17.4% isolates, respectively, while only 11.6% were resistant to 3–5 classes. Possible extensive drug resistance (pXDR) was found in 2.3% of isolates. High single resistance was observed for oxytetracycline (93%) and amoxicillin (91.9%), followed by ampicillin (89.5%), trimethoprim–sulfamethoxazole, and pefloxacin (88.4%), and tetracycline (84.9%). Most importantly, 89.6% of isolates were resistant to carbapenems. All the isolates were positive for the blaTEM gene. However, the blaSHV and blaCTX-M-2 genes were identified in two ESBL-non producer isolates. None of the isolates carried the blaCTX-M-1 gene. This study provided evidence of the existence of MDR and pXDR ESBL-Ec in frozen chicken meat in Bangladesh, which may pose a risk to human health if the meat is not properly cooked or pickled raw only. This emphasizes the importance of the implementation of good slaughtering and processing practices by the processors.
ARTICLE | doi:10.20944/preprints202103.0024.v1
Subject: Life Sciences, Biochemistry Keywords: Methicillin-resistant Staphylococcus aureus; Multidrug resistance; mecA gene; Frozen chicken meat; Bangladesh
Online: 1 March 2021 (13:56:33 CET)
Infections by methicillin-resistant Staphylococcus aureus (MRSA) are continuously expanding within the community. Chicken meat is usually contaminated by MRSA, and this contaminated chicken meat is an important source of foodborne infections in humans. In this study, a cross-sectional supershop survey was conducted to determine the prevalence and antimicrobial resistance pattern of MRSA in 113 domestic frozen chicken meat samples purchased from nine branded supershops available in five divisional megacities of Bangladesh. The study also focused on the determination of methicillin resistance gene in MRSA isolates. S. aureus was identified by standard culture-based and molecular methods, and subjected to antimicrobial susceptibility testing. MRSA was screened by cefoxitin disk diffusion test. Methicillin resistance gene was identified by PCR. Of samples, 54.9% were positive for S. aureus, and, of these, 37.1% isolates were identified as MRSA. All the isolates were multidrug resistant (MDR): 52.2% were resistant to 6−8 antimicrobial classes, and 47.8% isolates to 9−12 classes. Three (3.2%) isolates of S. aureus were possible extensively drug resistant. The highest rates of resistance were observed against cefoxitin (100%), followed by nalidixic acid, ampicillin and oxacillin (97.7%), colistin (91.3%), amoxicillin-clavulanic acid and amoxicillin (87%), penicillin-G and cloxacillin (82.6%), oxytetracycline (78.3%) and cefixime (73.9%). Screening of methicillin resistance gene revealed that 43.5% isolates of MRSA were positive for mecA gene. The high prevalence of MDR MRSA in frozen chicken meat samples in this study emphasizes the need for better sanitary education of food handlers in hygienic practices focusing on their potential role as reservoirs and spreaders of MRSA.
ARTICLE | doi:10.20944/preprints202012.0254.v1
Online: 10 December 2020 (12:27:31 CET)
Background: There is an upsurge in the consumption of chicken meat leading to a high influx of imported frozen chicken parts into the Ghanaian markets with little information on their microbial qualities. This study examined the microbial quality of imported frozen chicken parts from three major import countries (USA, the Netherlands and Brazil) into the Kumasi Metropolis. Methods: A total of 45 chicken meat parts of 15 thighs, wings and backs from wholesale cold stores market in the Kumasi Metropolis were randomly sampled for laboratory examinations. A ten-fold serial dilution was performed on each homogenized chicken parts to determine microbiological quality using Plate Count Agar , MacConkey Agar (MCA), Mannitol Salt Agar (MSA) and Desoxycholate Citrate Agar (DCA) for the total viable count (TVC), total coliform count (TCC), Staphylococcus and Salmonella spp counts respectively incubated at 37oC for 48 hours. Sabouraud Dextrose Agar (SDA) was used for fungal counts. We identified bacterial and fungal isolates using appropriate laboratory and biochemical tests. Descriptive data analysis was carried using SPSS-IBM version 16. Results: Mean TVCs of 5.93, 5.98 and 6.14 log10cfu/g were recorded for frozen chicken meats from the USA, the Netherlands and Brazil respectively. Means TCCs of 6.14, 5.93 and 5.98 log10cfu/g were obtained for chicken meats from Brazil, USA and the Netherlands respectively. Staphylococcus spp. (35.4%), E. coli (26.2%), Salmonella spp. (24.6%), and Klebsiella spp. (13.8%) were isolated with Aspergillus spp (33.3%), Rhizopus spp (27.3%), Penicillin spp (24.2%), and Cladosporium spp (15.2%). Chicken thighs, backs and wings recorded 46.2%, 29.2% and 24.6% bacterial contaminants in this order. Bacterial isolates of 49.2%, 28.8% and 22.0% were recorded in frozen chicken meat products from Brazil, the Netherlands USA respectively. Conclusion: The results suggest that imported frozen chicken meats into the Ghanaian market have moderate quality with potential pathogens such as E. coli and Salmonella spp.
TECHNICAL NOTE | doi:10.20944/preprints202009.0683.v1
Subject: Chemistry, Analytical Chemistry Keywords: DNA extraction; normal; patients; protocol; frozen blood
Online: 28 September 2020 (10:52:59 CEST)
Herein, we describe a highly efficient, non-complicated, and non-organic procedure to overcome the negative effect of chemotherapeutic drugs on the quality of the extracted DNA by applying several modulations in cell washing, suspension, and lysis of cells treated with these drugs. In this protocol, 500µl were extracted from patients who received systemic sessions of chemotherapy. The validity of this protocol for digestion with restriction endonucleases and both conventional and real-time polymerase chain reaction were tested. This protocol proved obvious purity (1.8±0.02 and 2.1±1.2, for A260/280 and A260/230, respectively) and adequate yields (10±2.24) µg/100 ml. The positive results of validation experiments proved the validity of the extracted DNA for downstream applications of molecular biology. In addition to the proven efficiency of this protocol to extract DNA from normal samples, its validity was also confirmed from patients who were exposed to chemotherapy. This entails a novel approach to extract a molecular biology grade DNA without having inhibitors against enzymes used in digestion, amplification, and subsequent sequencing even after the systemic sessions with several doses of chemotherapy.
ARTICLE | doi:10.20944/preprints202105.0757.v1
Subject: Life Sciences, Biochemistry Keywords: Ovine; Caprine; Cheese whey; Ultrafiltration; Frozen yoghurt; Inulin
Online: 31 May 2021 (12:00:15 CEST)
The objective of this work was the use of goat and sheep liquid whey concentrates (LWCs) produced by ultrafiltration (UF) for the manufacture of frozen yoghurts with or without different concentrations of inulin. In a first step, natural yoghurts using only goat and sheep LWCs as raw material were obtained. One day after production, these yoghurts were used to produce frozen yoghurts with different concentrations of added inulin. The physicochemical charateristics of ewe´s and goat´s yoghurts were significantly different regarding dry matter, protein, fat and minerals. Ewe´s yoghurts were solids while goat´s yoghurts behaved as a viscous liquid. Frozen yoghurts with different levels of inulin addition also presented significant differences concerning physicochemical and microbiological characteristics. Overrun was similar for all formulations except for the one produced with ewe’s LWC containing 5.0% inulin, which presented a significantly higher value. Higher meltdown rates in goat’s whey frozen yoghurts were observed. The survival rates of lactic acid bacteria were lower than data reported for similar products. Concerning sensory acceptance, both products showed encouraging results. It can be considered that the production of frozen yoghurts by using LWCs as main ingredient can be an interesting option to broaden the product’s portfolio of small/medium scale dairy producers.
ARTICLE | doi:10.20944/preprints202101.0510.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Frozen-Thawed Embryo Transfer; Birthweight; Endometrial Preparation; Ovulatory Cycle; Artificial Cycle
Online: 25 January 2021 (15:06:40 CET)
Background: It is unknown whether prolonged artificial hormonal environment during early fetal development affects the birthweight of singletons born after frozen-thawed embryo transfer (FET). Methods: A retrospective observational study included singleton births>22 weeks of gestation obtained after FET between 2013-2019 after endometrial preparation with ovulatory cycle (OC) or artificial cycle (AC). Our primary objective was to compare birthweight of singletons after FET between endometrial preparation by OC or AC. Secondary objectives included prolonged pregnancies, high birthweight, low birthweight, SGA and LGA rates. Multivariate analyses were performed considering potential confounding factors. Results: Among 198 singleton live births after FET, 112 were obtained with OC and 86 with AC. Prolonged pregnancies rate was higher in AC (25.6% vs. 7.1%, respectively, p=0.001). Mean birthweight was higher (+219g) in AC (3386g vs. 3167g, p=0.01; adjusted-p=0.052), as well as the rate of babies exceeding 4000g (16.3% vs. 2.7%, p=0.03, adjusted-p=0.015). The rate of babies <2500g was lower in AC (3.5% vs. 11.6%, respectively, p=0.050, adjusted-p=0.049). Conclusions: Since OC does not strain the chances of pregnancy and in the incomplete knowledge of the consequences of neonatal overweight on the future health of children, OC preparation could be advocated as first-line endometrial preparation in FET.
ARTICLE | doi:10.20944/preprints202008.0285.v1
Subject: Life Sciences, Virology Keywords: FMDV; Egypt; full-genome sequencing; interserotypic recombination; vaccine composition analysis; frozen evolution
Online: 12 August 2020 (11:56:33 CEST)
In addition to the risk of vaccine failure caused by strain mismatch, the production of inactivated FMD vaccines is dangerous if adequate biosafety cannot be maintained. Using a high-throughput sequencing protocol optimized for short nucleic acid fragments, the composition of a local inactivated vaccine was analyzed in depth. The serotype O strain identified in the vaccine was genetically identical to viruses found in recent FMD outbreaks in Egypt.
CONCEPT PAPER | doi:10.20944/preprints201705.0121.v1
Subject: Biology, Other Keywords: Genetic code evolution; frozen accident; error minimization; stereochemcial theory; evolution of translation
Online: 16 May 2017 (12:52:10 CEST)
Nearly 50 years ago, Francis Crick propounded the frozen accident scenario for the evolution of the genetic code along with the hypothesis that the early translation system consisted primarily of RNA. Under the frozen accident perspective, the code is universal among modern life forms because any change in codon assignment would be highly deleterious. The frozen accident can be considered the default theory of code evolution because it does not imply any specific interactions between amino acids and the cognate codons or anticodons, or any particular properties of the code. The subsequent 49 years of code studies have elucidated notable features of the standard code, such as high robustness to errors, but failed to develop a compelling explanation for codon assignments. In particular, stereochemical affinity between amino acids and the cognate codons or anticodons does not seem to account for the origin and evolution of the code. Here I expand Crick’s hypothesis on RNA-only translation system by presenting evidence that this early translation already attained high fidelity that allowed protein evolution. I outline an experimentally testable scenario for the evolution of the code that combines a distinct version of the stereochemical hypothesis, in which amino acids are recognized via unique sites in the tertiary structure of proto-tRNAs, rather than by anticodons, expansion of the code via proto-tRNA duplication and the frozen accident.
ARTICLE | doi:10.20944/preprints201701.0110.v1
Subject: Mathematics & Computer Science, Applied Mathematics Keywords: systems of nonlinear equations; nonlinear preconditioners; multi-step iterative methods; Frozen Jacobian
Online: 25 January 2017 (04:13:19 CET)
The study of different forms of preconditioners for solving a system of nonlinear equations, by using Newton’s method, is presented. The preconditioners provide numerical stability and rapid convergence with reasonable computation cost, whenever chosen accurately. Different families of iterative methods can be constructed by using a different kind of preconditioners. The multi-step iterative method consists of a base method and multi-step part. The convergence order of base method is quadratic and each multi-step add an additive factor of one in the previously achieved convergence order. Hence the convergence of order of an m-step iterative method is m + 1. Numerical simulations confirm the claimed convergence order by calculating the computational order of convergence. Finally, the numerical results clearly show the benefit of preconditioning for solving system of nonlinear equations.
ARTICLE | doi:10.20944/preprints201608.0003.v1
Subject: Earth Sciences, Environmental Sciences Keywords: seasonally frozen soil; frost heave; soil moisture content; soil type; freezing depth; soil porosity
Online: 1 August 2016 (09:47:52 CEST)
Frost heave, which is the volumetric expansion of frozen soil, has great ecological significance, since it creates water storage spaces in soils at the beginning of the growing season in cold temperate forests. To understand the characteristics of frost heave in seasonally frozen soil and the factors that impact its extent, we investigated the frost heave rates of forest soil from different depths and with different soil moisture contents, using both lab-based simulation and in situ measurement in a broadleaved Korean pine forest in the Changbai Mountains (northeastern China). We found that frost heave was mainly affected by soil moisture content, soil type, and gravitational pressure. Frost heave rate increased linearly with soil moisture content, and for each 100% increase in soil moisture content, the frost heave rate increased by 41.6% (loam, upper layer), 17.2% (albic soil, middle layer), and 4.6% (loess, lower layer). Under the same soil moisture content, the frost heave rate of loam was highest, whereas that of loess was lowest, and the frost heave of the uppermost 15 cm, which is the biologically enriched layer, accounted for ~55% of the frost heave. As a result, we determined the empirical relationship between frost heave and freezing depth, which is important for interpreting the effects of frost heave on increases in the storage space of forest soils and for calculating changes in soil porosity.
REVIEW | doi:10.20944/preprints202009.0162.v2
Subject: Biology, Anatomy & Morphology Keywords: amyloids; frozen accident; genetic code; hydrogels; liquid-liquid phase separation; mRNA; polyglycine; rRNA; ribosomes; translational fidelity; tRNA
Online: 21 October 2020 (10:48:18 CEST)
The genetic code evolved by parallel tracks of chaotic and ordered processes. Liquid-liquid phase separation (hydrogels), a chaotic process, constructs diverse membraneless compartments within cells, resulting in regulated hydration and sequestration and concentration of reaction components. Hydrogels relate to chaotic amyloid fiber production. We propose that polyglycine and related hydrogels (i.e. GADV; G is glycine), phase separations, membraneless droplets and amyloid accretions organized protocell domains to drive the earliest evolution of the genetic code and the pre-life to cellular life transition. By contrast, evolution of tRNA, tRNAomes, aminoacyl-tRNA synthetases and translation systems followed highly ordered and systematic pathways, described by well-defined mechanisms and rules. The pathway of evolution of aminoacyl-tRNA synthetases, which tracked evolution of the genetic code, is clarified. Hydrogels and amyloids form a chaotic component, therefore, that complemented otherwise systematic processes. We describe with detail a pre-life world in which hydrogels and amyloids provided the selections of the first life.
ARTICLE | doi:10.20944/preprints202008.0232.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: Brassica yellows virus (BrYV); Myzus persicae; transgenic plants with BrYV full length genome; frozen BrYV infected plants; acquisition and transmission
Online: 10 August 2020 (03:34:00 CEST)
Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which occurs widely and mostly damage Brassicaceae plants in East Asia. Since BrYV could not be transmitted mechanically, an insect transmission method is required for further virus research. Here, a reliable and unrestricted method was described, in which non-viruliferous aphids (Myzus persicae) acquired BrYV from transgenic Arabidopsis thaliana plants with virus full length genome germinated from seeds and frozen infected leaves were used to transmit the virus to healthy plants, and there was no significant difference in acquisition rate though transmission rate from frozen infected leaves was somehow lower compared to fresh infected leaves. This novel simple method could be applied to preservation of virus inocula, evaluation of variety resistance to BrYV, biological research on interaction among BrYV, aphid and host, which also provide a new idea on establishing a basic method using virus genomic transgenic plants or frozen infected leaves for other poleroviruses research.