Alkaline Lysis Method Research Articles

Antibiotic Resistant and Plasmid Borne Bacteria Associated with Locally Produced Honey in Enugu State, Nigeria

Aim: The aim of this study is to isolate and identify bacteria contaminants present in the honey samples sold in Ogbete market and detect the presence of imipenem resistance genes in the isolates. Place and Duration of Study: A total of four samples were collected from Ogbete market at Zenith bank, Peace mass, Wheelbarrow and Peace park stations between the early hours of the morning (8-9am) within the month of April, 2023. Methodology: Microbiological analysis was carried out on the samples, including antimicrobial susceptibility test using disc diffusion method., after which Molecular analysis involving DNA extraction, PCR and Plasmid Profiling, were carried out. Results: The total mean heterotrophic bacterial count ranged from 0.03±2.5×103cfu/ml to 22.1±25.5×103cfu/ml. The highest total mean heterotrophic bacterial count was recorded in Zenith bank while the lowest was in Peace mass. The bacteria isolated and characterized through morphological and biochemical tests include Escherichia coli (45%) Klebsiella spp. (41%), Staphylococcus spp. (9%) and Streptococcus spp. (5%). Escherichia coli had the highest frequency of occurrence at 45% while Streptococcus spp. had the lowest at 5%. The sensitivity test showed that the isolates with the highest frequency of occurrence, Escherichia coli and Klebsiella spp. posed a high resistance to Imipenem and Nalidixic at 80% while the least resistance was in Levofloxacin at 10% from these two isolates. The gene of interest, BlaIMP was found after PCR assay. Plasmid DNA was further carried out using the alkaline lysis method using the Zymo research kit. All isolates possessed a single plasmid with molecular weight of 23.1kbp. Conclusion: These isolates may undergo horizontal gene transfer and confer resistance to other microorganisms. Thus, there is need for the development of proper measures to counter bacterial contamination of locally produced honey as this poses a threat to public health.

Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran: A descriptive-analytical study

Objective: To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection. Methods: In this study, 100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational- therapeutic hospitals and were identified using standard microbiological tests. Then, the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method. The bacterial DNAs were extracted by the alkaline lysis method. Finally, the presence of exoU and exoY genes was evaluated by the PCR test. Results: In this study, 47%, 72%, 29%, 39%, 40%, and 44% of the isolates were non-susceptible to piperacillin, aztreonam, ceftazidime, imipenem, tobramycin, and ciprofloxacin, respectively. In addition, 95% and 93% of the clinical isolates carried the exoU and exoY genes. Blood and fecal isolates had both virulence genes, while only one wound isolate had neither genes. Meanwhile, all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene. Also, 88 isolates simultaneously had both exoU and exoY genes. Conclusions: High prevalence of exoU and exoY genes in this region indicates a significant role of type III secretion system in pathogenesis of Pseudomonas aeruginosa. The type III secretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Antimicrobial resistance patterns, virulence genes, and biofilm formation in enterococci strains collected from different sources

BackgroundCurrently, antibiotic-resistant strains of Enterococcus are considered to be one of the critical health challenges globally. This study aimed to investigate the antibiotic susceptibility pattern, biofilm formation capacity, and virulence genes of enterococci isolated from different sources.MethodsIn this cross-sectional study, environmental and fecal samples were collected from the hospital environment, volunteers, and hospital staff from October 2018 to August 2019. The isolates were identified by morphological and biochemical tests (gram staining, catalase, bile resistance, esculin hydrolysis, carbohydrate fermentation, growth in 6.5% NaCl, Pyrrolidonyl arylamidase, arginine dehydrolase), and PCR for ddl gene. An antimicrobial susceptibility test was performed by the standard disk agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quantitative microplate assays were used to assess biofilm production. The bacterial DNAs were extracted by alkaline lysis method and polymerase chain reaction technique was used detect the esp, ace, and efaA virulence genes.ResultsOut of 145 isolates, 84 (57.9%) were identified as E. faecalis and 61 (42.1%) as E. faecium. Resistance to kanamycin and quinupristin-dalfopristin was 82.1% (69/84) and 85.7% (72/84), respectively, in E. faecalis isolates. Out of 61 E. faecalis isolates, 38 (62.4%) were resistant to kanamycin. Among the E. faecalis isolates, esp was the most dominant virulence gene (73.80%), followed by efaA, and ace, which were detected in 60.71%, and 30.95% isolates, respectively. In total, 68.27% of the strains were biofilm producers. Further, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm biomass.ConclusionsAccording to the findings of our study, enterococci strains isolated from different samples possess distinctive patterns of virulence genes. The esp, ace, and efaA genes were more prevalent among E. faecalis than E. faecium. Besides, the high level antibiotic resistance of normal flora and environmental enterococci strains is alarming the researchers.

The 60 nm gold nanoparticles improve qPCR amplification efficiency through specific palindromic sequences (GGATCC or ACCGGT) in primers

BackgroundPolymerase chain reaction (PCR) technology and quantitative real-time PCR (qPCR) technology are widely used in clinical diagnosis and research, but amplification efficiency and sensitivity are still key problems for researchers. An increasing number of reports show that gold nanoparticles (AuNPs) can be used to improve the sensitivity and amplification efficiency of PCR. Here, we found that 60 nm gold nanoparticles with a positive charge (60 nm- Au+) can enhance the amplification efficiency of qPCR. MethodsMouse DNA was extracted by the alkaline lysis method. Primer 5.0 software was used to design primers and mutation primers, and the DNA fragments were obtained by the method of synthesizing plasmids. The qPCR was applied to amplify target gene fragments. ResultsThe amplification efficiency of qPCR was improved by about 1.828 times in the experimental group with 60 nm- Au+ compared with the control group without 60 nm- Au+. The primer pair contains a specific palindromic sequence (GGATCC or ACCGGT). And 60 nm Au+ did not enhance the amplification efficiency of qPCR when the above primer was mutated. ConclusionsThe primers contain special palindrome sequences (GGATCC or ACCGGT) with 60 nm- Au+ can enhance the amplification efficiency of qPCR. Therefore, it suggests a more in-depth understanding of the mechanism and function of gold nanoparticles and primer sequences. This study has presented some implications for gold nanoparticles application in the development of qPCR technology.

Isolation of Plasmid Dna from Bacteria Associated With Ulcerative Disease in <i>Channa punctatus</i>

Ulcerative disease has been prevalent particularly in snake head and catfishes. A number of virulent bacteria are associated with this disease. A variety of important properties viz., virulent determinants, antimicrobial resistance, etc. of the pathogenic bacteria that enabled them to thrive better in the adverse environment, have been proven to be plasmid encoded. Keeping in mind the importance of plasmid DNA, this study has been undertaken. Plasmid DNA was isolated by alkaline lysis method from the cultured bacteria associated with ulcerative disease of snake head, Channa punctatus. Isolation of plasmid DNA followed by agarose gel electrophoresis analysis have revealed the presence of a single plasmid DNA in the cultured microbe which comigrated with 23 kb DNA fragment of the lambda DNA digested by Hind III. The cultured microbe was characterised as: Gram-negative motile rod, catalase-positive, oxidase-negative, indole-negative, non-HS producing, lactose fermenting, acid and gas produced from glucose, methyl red-negative, Voges Prauskauer-positive and urea negative. It seems from the above mentioned biochemical as well as morphological characteristics that the cultured microbe was very similar to Enterobacter and Citrobacter.

An improved DNA extraction method for detecting Bacillus subtilis spores in spiked foods and beverages

Bacillus and Paenibacillus spp. are essential aerobic spoilage bacteria in various food industry sectors. Spoilage from microorganisms occurs at many points throughout food production systems. Due to their complex wall structures, spores can resist heat, radiation, chemical agents, and enzymatic treatments. An alkaline lysis and mechanical disruption combination method was developed and evaluated to counter this. This combination method effectively improved DNA extraction from B. subtilis spore cells spiked into food (solid) and beverages (liquid milk and coffee) at concentrations down to 102 CFU/mL or g when spiked into food matrices and drinks. Released DNA recoveries were 27 % and 25 % for potato salad and 38 % and 36 % for whole corn spiked at 106 and 103 CFU/mL concentrations. Conversely, there was a low recovery for wheat flour (10 % and 8.8 %) and milk powders (12 % and 25 %) at 106 and 103 CFU/mL spiked concentrations. The combination method provides rapid, specific, reliable, and accurate signature sequences identification for the detection and presence confirmation of psychrophilic and psychrotolerant spoilage spore cells, improving food spoilage assessments and food control applications.

Development and validation of isothermal polymerase spiral reaction assay for the specific authentication of goat (Capra hircus) meat

Goat meat (chevon) is one of the costlier meats and closely resembles other red meat species mostly the sheep meat (mutton). In order to establish authenticity of chevon and confirm mislabelling there is a need for a reliable laboratory test. Polymerase spiral reaction (PSR) assay is one of the recent DNA based isothermal tests available for molecular analysis mostly microorganisms. Present study describes development and validation of a novel PSR for the detection of chevon for the first time. The goat specific PSR makes use of a pair of chevon-specific primers targeted against the mitochondrial DNA and test could be read based on the visual colour change after the addition of SYBR Green I dye to the amplified product. The possibility of cross-amplification was ruled out by testing related meat species ( buffalo, cattle, chicken, pig, sheep, and ostrich) and results of PSR were further confirmed by fluorometry and agarose gel electrophoresis. PSR could be undertaken at isothermal temperature of 62 °C temperature for 60 min with the limit of detection of 0.5 ng DNA and detection of meat admixing of 0.1%. The PSR was found efficient in the detection of not only raw meat but also meat heated to 121 °C for 30 min. When combined with the alkaline lysis method of DNA extraction, the goat-specific PSR takes only 90 min of sample analysis time and could be used for the rapid analysis of field samples.

Optimization of High Concentration Plasmid DNA for Use in COVID-19 mRNA Vaccine Development: Comparison of Between Alkaline Lysis Method and Commercial Kit Results

While forming the stable IVT mRNA molecule, high concentration and purity plasmid DNA must be obtained to ligase the ORF antigen sequence initially copied from the plasmid DNA with the UTR regions. In this study, in the stage of creating the mRNA molecule, which is the first step of the COVID-19 mRNA vaccine, comparison and optimization of the pDNA containing the ORF target antigen sequence were performed as a result of isolation with alkaline lysis method and commercial kit. Plasmid DNA bacteria containing the target antigen ORF sequence were grown under appropriate conditions. Plasmid DNA was isolated by commercial kit and alkaline lysis method from bacterial cultures stopped at different OD600 nm values (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5). After the obtained pDNAs were visualized on an agarose gel, their purity and concentration were measured by spectroscopic measurement. After the stab culture is resuscitated in SOC medium, bands are formed in a single form after isolation with the kit, and in multiple forms (linear, supercoiled, circular) after pDNA isolation by alkaline lysis method. The ideal OD600 nm for both methods was 0.3-0.4. As a result of isolation with the kit, higher purity on the contrary low concentration pDNA was obtained. The ideal OD600 nm value is a critical parameter that affects the concentration and purity of pDNA. The alkaline lysis method is a cheap and powerful technique that can be used as an alternative for mRNA vaccine development compared to kit isolation.

Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA

Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.

Dissemination of Quinupristin-Dalfopristin and Linezolid resistance genes among hospital environmental and healthy volunteer fecal isolates of Enterococcus faecalis and Enterococcus faecium.

Streptogramins and linezolid are important in the treatment of infections caused by vancomycin-resistant enterococci. Then, we aimed to evaluate the resistance rates against these drugs and the prevalence of genes involved in hospital environmental and fecal normal-flora isolates of Enterococcus faecalis and Enterococcus faecium. The strains were isolated from the stool samples and hospital environments by culturing on M-Enterococcus (ME) agar, and identified by phenotypic and genotypic microbiological tests. The disk agar diffusion method was used to identify the antimicrobial susceptibility pattern of the isolates. The genomic DNA extraction was done by the alkaline lysis method, and the PCR test was used to detect the resistance genes. A total of 145 enterococci isolates were taken, from which 84 (57.9%) isolates were detected as E. faecalis and 61 (42.06%) isolates were E. faecium. Moreover, 70 (83.33), 4 (4.76%), 1 (1.19%), and 40 (47.61%) isolates of E. faecalis and 20 (32.78%), 1 (1.63%), 4 (6.55%), and 26 (42.62%) E. faecium isolates were resistant against quinupristin-dalfopristin, linezolid, vancomycin, and erythromycin, respectively. Also, 112 (77.24%), 50 (34.48%), 39 (26.89%), 27 (18.62%), 19 (13.1%), 4 (2.75%), and 1 (0.68%) isolates were contained LsaA, vatD, vgbB, vatE, cfr, lsaE and optrA genes, respectively. None of the isolates carried the vgbA gene. High-level streptogramin resistance rate and high prevalence of resistance genes in enterococci isolated from the stool of healthy persons and the hospital environment indicates the importance of possible transmission of resistance genes from these isolates to clinical ones.

BACTERIOLOGICAL QUALITY OF INDOOR AIR ENVIRONMENT IN PRIVATE AND GOVERNMENT OWNED ABATTOIRS IN BENIN CITY, NIGERIA

The airborne bacterial load of two abattoirs, private and government abattoirs in Benin City was studied using the settled plate methods. The air samples were collected during and after slaughtering operations in the two abattoirs monthly for a period of six months, between October, 2011 and March, 2012. The temperature and relative humidity of the two abattoirs were determined during each sampling time. The plasmid DNA profile of the isolated airborne bacterial was determined using the alkaline lysis method. The temperature reading ranged between 25 0C and 34 0C in the private abattoir and between 26 0C and 35 0C in the government abattoir. The relative humidity reading ranged between 46 % and 67 % in the private abattoir and between 34 % and 65 % in the government abattoir. The mean airborne bacterial counts on nutrient agar and blood agar in the private abattoir ranged from 17.55 x 102 ± 3.77cfu/m3 to 108.0 x 102 ± 10.35cfu/m3 during slaughtering and 12.84 x 102 ± 1.51 cfu/m3 to 49.71 x 102 ± 3.61 cfu/m3 after slaughtering. The mean airborne bacterial counts on nutrient agar and blood agar in the government abattoir ranged from 24.63 x 102 ± 3.75cfu/m3 to 58.30 x 102 ± 6.49 cfu/m3 during slaughtering and 15.62 x 102 ± 2.97 cfu/m3 to 114.13 x 102 ± 19.75 cfu/m3 after slaughtering. The mean airborne bacterial counts grown on eosine methylene blue agar in the private abattoir ranged from 1.057 x 102 ± 2.58 cfu/m3 to 22.54 x 102 ± 5.70 cfu/m3 during slaughtering and 2.44 x 102 ± 1.41 cfu/m3 to 9.87 x 102 ± 1.41 cfu/m3 after slaughtering. The mean airborne bacterial counts grown on eosine methylene blue agar in the government abattoir ranged from 1.05 x 102 ± 5.62 cfu/m3 to 24.40 x 102 ± 4.66 cfu/m3 during slaughtering and 1.06 x 102 ± 4.24 cfu/m3 to 21.08 x 102 ± 3.94 cfu/m3 after slaughtering. The result revealed the isolation of seven airborne bacterial isolates which include Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus sp., Bacillus sp., Pseudomonas sp., Klebsiella sp., and Escherichia coli. The frequency of occurrence and distribution of the airborne bacterial isolates revealed Staphylococcus. aureus (48.75%) and Bacillus sp. (28.68%) as the most frequently isolated airborne bacterial isolates in the private abattoir, while S. aureus (31.95%) and Bacillus sp. (41.93%) were also recorded as the most frequently isolated airborne bacterial isolates in the government abattoir. The statistical analysis showed no significant difference between the airborne bacterial counts recorded during and after slaughtering in the two abattoirs. The result of the plasmid determination revealed that the airborne bacterial isolates harboured plasmid sizes which ranged between 1.5 kbs to 25.2 kbs. The result revealed that, the quality of air in the abattoirs was greatly influenced by the population and type of airborne bacterial isolates, which play significant role in the level of hygiene in meat processing plants. The type and population of airborne bacterial isolates in the indoor air of the two abattoirs are of great concern, which serves as index of quality of air in the environment. Hence, great measures are taken to checkmate this menace caused by the poor hygienic systems in the abattoirs

Antibiotic Susceptibility and Plasmid Profile of Multidrug resistant Uropathogenic Serratia marcescens

Objectives: Genetic elements such as plasmids play a role in spreading multi-antibiotic resistance, a global threat. Therefore, the purpose of this study was to investigate the antibiotic susceptibility and plasmid profiles of multidrug-resistant (MDR) uropathogenic Serratia marcescens. Methods: Fifty Serratia marcescens isolates were obtained from urine samples of patients presenting with urinary tract infection (UTI) in Southeastern Nigeria. Bacteria samples were identified via their characteristic red pigmentation and other biochemical tests. The disc diffusion method was used for antibiotic susceptibility testing. Plasmids were extracted using the alkaline lysis method, and electrophoresis was done on a 1% agarose gel. Extracted DNA plasmids were visualized using the ultraviolet light illuminator and a photo documentation system. Plasmid curing was carried out using acridine orange. Results: Antibiotic susceptibility testing of isolates revealed 41 (82%) were resistant to one or more antibiotics tested, and 8 (16%) isolates exhibited resistance to three or more antibiotics (MDR). Of the eight MDR isolates, five (63%) had plasmids with molecular weights ranging from 33 kb to 58 kb. One isolate (S9) was cured and became sensitive to ceftriaxone, but not cefuroxime or ceftazidime. Notably, one isolate (S23), initially sensitive to ciprofloxacin, became resistant post-plasmid curing. Conclusion: The findings show that most resistant bacteria containing plasmids were immune to curing as they remained resistant to antibiotics after curing. This result confirms a concern about the growing presence of MDR plasmids of S. marcescens in healthcare facilities in Southeastern Nigeria. J Microbiol Infect Dis 2022; 12(1):12-18.

Antibiotic Resistance and Transfer of Extended Spectrum Beta Lactamase (ESBL) Resistant Plasmids in Pseudomonas aeruginosa Isolates from Hospitalized Patients

The study was undertaken to determine antibiotic resistance, ESBL production and transfer of resistant plasmids of Pseudomonas aeruginosa. Clinical samples of urine, wound and blood were screened for Pseudomonas aeruginosa following standard microbiological procedures. Isolates of Pseudomonas aeruginosa were identified in a total of 110 (18.3%) samples with higher prevalence observed in wound (43.6%) and blood (40.0%) samples. Identified isolates were profiled for antimicrobial susceptibility to a total of 10 antibiotics. Plasmid DNA extraction was carried out by the alkaline lysis method. Double disc synergy test (phenotypic) and PCR (genotypic) techniques were used to determine ESBL production from selected organisms while transfer of resistant genes was assessed by conjugation using broth mating procedures. Results showed high rates of resistance among organisms tested using antipseudomonal drugs. Seventy-seven (70.0%) of the isolates were multi drug resistant (MDR), out of which 26 (33.8%) and 4 (15.4%) produced ESBL by phenotypic and genotypic methods, respectively. Plasmid profile analysis revealed that all the 77 MDR isolates harboured plasmids of &gt; I.5 kb. Four ESBL producing isolates selected could transfer their resistance plasmid to a recipient E. coli strain K-12. The therapeutic challenge observed calls for an effective antimicrobial stewardship program.

Recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a threatened species

This study addresses the use of recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a snake species threatened due to over–exploitation and habitat destruction. For its conservation, an efficient species identification method is urgently neededto fight against illegal capture and trade. Fourteen individuals representing 12 snake species (including D. acutus and other snake species) were collected from mountainous regions in Southern China. Genomic DNA was extracted within five minutes by a modified alkaline lysis method. Species–specific primers for recombinase polymerase amplification (RPA) were designed based on the sequences of cytochrome C oxidase subunit I (COI) barcode region, and an optimized RPA assay system was set up. Specificity and sensitivity of the assay were checked, and the assay was validated by identifying 10 commercial Qi She crude drug samples derived from D. acutus. Under optimized RPA conditions, a distinct single band of 354 bp was amplified only for D. acutus but not for the related snake species. The entire procedure can be completed in 30 min at room temperature. Commercial Qi She crude drug identification validated effectiveness of the established assay system. Using a recombinase polymerase amplification (RPA) assay with rapid DNA extraction, we established an on–spot D. acutus identification method with good specificity and sensitivity. This method could become an efficient tool for rigorous supervision of illegal D. acutus capture and trade.

High prevalence of blaVEB , blaGES and blaPER genes in beta-lactam resistant clinical isolates of Pseudomonas aeruginosa.

The increased prevalence of β-lactamase is one of the main factors in resistance to β-lactams in Pseudomonas aeruginosa. This study aimed to investigate the prevalence of blaVEB, blaPER, and blaGES genes in β-lactam-resistant P. aeruginosa. We collected 100 non-duplicated clinical isolates of P. aeruginosa and identified them by standard tests. Using disk agar diffusion test, we detected the β-lactam-resistant isolates and extracted the DNAs of the isolates by alkaline lysis method. Then, the prevalence of blaVEB, blaPER and blaGES genes were detected by PCR method. The results were assessed by SPSS 21 software and Chi-square test. Out of 100 isolates, 43% were detected as resistant against at least one of the beta-lactams tested. Piperacillin-tazobactam was the most effective antibiotic, while 39% and 37% of the isolates were resistant to aztreonam and meropenem, respectively. A significant relationship was observed between the resistance to tested antibiotics and the presence of blaVEB, blaGES, and blaPER genes. Among 43 isolates that were resistant to at least one of the tested β-lactams, 93.02%, 83.72%, and 81.39% of them carried blaVEB, blaGES, and blaPER genes, respectively. According to this study and due to high prevalence of β-lactam resistance genes, it is better to check the level of antibiotic resistance and resistance genes for better management of patients with infection caused by this bacterium. Also, high prevalence of class A β-lactamases indicates the significant role of these enzymes in emerging resistance to beta-lactams.

Effect of Post Treatment with Brine and Steam on Phenotypic Resistant &amp;lt;i&amp;gt;Salmonella&amp;lt;/i&amp;gt; Isolates from &amp;lt;i&amp;gt;Pentaclethra macrophylla&amp;lt;/i&amp;gt;

Food contaminated with antibiotic resistant bacteria pathogens is a major threat to public health. Apart from infecting man they serve as reservoirs of genes for antimicrobial resistance and they easily transfer the resistant genes to both related and unrelated bacterial species; hence the aim of the present study. The prevalence, antibiotic sensitivity pattern and plasmid profile of <i>Salmonella</i> spp isolates from ready-to-eat ugba (<i>Pentaclethra macrophylla</i>) samples vended in various markets in Enugu State, Nigeria was investigated. The samples were further examined to determine the effect of post treatment with brine and steam respectively. A total of 40ugba (<i>P. macrophylla</i>) samples were obtained from different food vendors in the selected markets in Enugu metropolis, Enugu state, Nigeria. Cultures were done on Salmonella-shigella agar and characterized by standard microbiological methods. Post treatment with brine involved rinsing 5g of <i>P. macrophyla</i> samples in three changes of brine solution while post treatment using steam was done by boiling5g of <i>P. macrophyla</i> samples for 10 mins to an internal temperature of 30°C, 60°C, 75°C and 90°C and subsequently cultured on Salmonella-Shigella agar by pour plate method. Antibiogram of the isolates were determined by using disc diffusion method and plasmid DNA was extracted using plasmid alkaline lysis method and separated by agarose gel electrophoresis. <i>Salmonella</i> spp. was recorded at high prevalence of 95%. The isolates showed varied resistance to different antibiotics; amoxicillin (50%), augmentin (100%), streptomycin (61%), septin (95%), gentamycin (45%) and chloramphenicol (68%). Multiple antibiotic resistant was observed in 26% of the isolates. Agarose gel electrophoresis showed that all the ten selected isolates had single plasmid bands of 20kb size. The results revealed relative similarities between the <i>Salmonella</i> isolates but possibility of five clones among the <i>Salmonella</i> isolates. At p<0.05 steam reduced the microbial load of isolates in <i>P. macrophyla</i> than in brine. The results from this study show that <i>Salmonella</i> spp. contamination of <i>P. macrophyla</i> was high in the study area and isolates were multidrug resistant; therefore food handlers should be properly educated and monitored to ensure compliance to proper food hygiene guidelines and <i>P. macrophyla</i> should be steamed prior to consumption to reduce the rate of infection.

Determination of Plasmid Profile Multiple Antibiotics Resistant Pseudomonas aeruginosa Isolated from Clinical Specimens in Healthcare Facilities across Anambra and Imo States Nigeria

The emergence and dissemination of antibiotic resistant plasmids of P. aeruginosa is posing a major public threat and huge concern in hospital facilities. This study was done across Anambra and Imo States with a total number of 100 P. aeruginosa isolates, 50 from each State to determine the plasmid profile of multidrug resistant isolates. Methods of re-identifying P. aeruginosa were based upon cultural methods coupled with biochemical tests. To study the susceptibility of these isolates using disk diffusion method, seven (7) antibiotics were used namely: Piperacillin–tazobactam (100/10 µg), Ceftazidime (30 µg), Amikacin (30 µg), Ciprofloxacin (5 µg), Cefuroxime (30 µg), Ceftriazone (30 µg) and Gentamicin (10 µg). Plasmid extraction was done using alkaline lysis method after growing isolates resistant to more than four antibiotics on a nutrient broth. Agarose gel electrophoresis of plasmid DNA was carried out on 2% agarose gel slab in 1X TAE buffer. The results show that, the nutrient agar at 37˚C aerobically, P. aeruginosa isolates were recovered, which produced greenish-yellow pigment colonies, oxidase was positive and negative for gram stain. In Anambra, the result showed 100% resistance to Cefuroxime, Ceftazidime and Ciprofloxacin, 90% and 86% for Ceftriaxone and Piperacillin-tazobactem, 48% and 40% to Gentamicin and Amikacin respectively. Whereas in Imo state, the result showed 100% resistance to Ceftazidime and Ciprofloxacin, 80% to Ceftriaxone, Cefuroxime and Piperacillin-tazobactem while the least resistance was seen in Amikacin, and Gentamicin. Plasmid size ranging from 100bp to &gt;1000bp was detected from most of the multidrug resistant isolates. Not all the isolates with multidrug resistance were found to possess plasmids. It can be seen from this study that multidrug resistance in P. aeruginosa is not strictly plasmid-dependent (mediated).

Alkaline lysis‐loop mediated isothermal amplification assay for rapid and on‐site authentication of buffalo (Bubalus bubalis) meat

AbstractIndia is the largest producer and exporter of buffalo (Bubalus bubalis) meat in the world. But there are restrictions on the slaughter of cattle (Bos indicus/Bos taurus) and export of beef (cattle meat) from the country. This led to the incidences of misrepresentation of beef and other low cost meat as buffalo meat during export and hence necessitates reliable techniques for species authentication of buffalo meat. Tests need to be rapid to ensure that export consignments are not held for a long time while meat samples are being authenticated. In the present study, a rapid assay for authentication of buffalo meat was developed by employing alkaline lysis (AL) method of DNA extraction and loop‐mediated isothermal amplification (LAMP) using novel primers designed to target mitochondrial D loop region. The results of the AL‐LAMP assay were visualized by SYBR Green I dye. Further, the LAMP amplification was confirmed by agarose gel electrophoresis and fluorometry. The developed AL‐LAMP assay was validated using raw, heat treated, and admixed/adulterated buffalo meat samples. The AL‐LAMP assay could be carried out with minimum instrumentation and hence, holds potential for use as on‐site assay for species authentication of buffalo meat.

Antibiotic Resistance and Plasmid Profile Analysis of Salmonella Enteritidis Isolated in Siberia and the Far East of Russia between 1990 and 2017.

Salmonella is one of the major causes of foodborne disease outbreaks globally. Specifically, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the major causes of zoonotic Salmonella infection in humans worldwide. In this study, we present data on antimicrobial resistance (AMR) and plasmid profiles of S. Enteritidis strains isolated from patients, food, and the environment in Siberia and the Far East of Russia obtained during Salmonella monitoring between 1990 and 2017. A total of 345 S. Enteritidis isolates were tested by the disk diffusion method with a set of 15 antibiotics using EUCAST breakpoints v. 10 and by plasmid profile analysis using the alkaline lysis method. The results have shown a substantial decrease in susceptibility to aminoglycosides and quinolones during the study period. No significant differences were found in the susceptibility of strains between regions as well as in the its correlation with different plasmid types of the pathogen. Several S. Enteritidis strains were found to be resistant to ampicillin, kanamycin, tetracycline, chloramphenicol, and cephalosporins. All tested S. Enteritidis strains were susceptible only to imipenem. In this study, we observed a relatively low level of AMR in S. Enteritidis strains isolated in Siberia and the Far East of Russia. Nevertheless, it is important to continue the molecular genetic monitoring and AMR surveillance of S. Enteritidis to track further increases in AMR using conventional phenotypic susceptibility testing and by introducing whole-genome sequencing to identify AMR mechanisms.

A LAMP-based microfluidic module for rapid detection of pathogen in cryptococcal meningitis

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on microfluidic detection for CM diagnosis is still limited. We developed a multifunctional microfluidic module that integrated the pathogen enrichment and on-chip nucleic acid extraction. The module adopted a simple filtration membrane to effectively capture Cryptococcus cells and simplify the process, and combined lyticase digestion, alkaline lysis and heating methods to optimize the strategy to achieve nucleic acid extraction. The entire process was operated in the module, which reduced the exposure risk of directly processing cryptococcal samples. A portable one-pot lyophilized LAMP reagent bead with no temperature limit was developed, which improved the flexibility of operation. This module did not require any additional instrument, and is promising to develop a simple, rapid, and efficient approach to realize the “sample in and answer out” detection of real CSF samples. This microfluidic module had practical prospects and is expected to replace LFA for efficacy evaluation and follow-up in the middle and late stages of CM treatment, and could be used as an auxiliary method to confirm cases with questionable LFA results in the early diagnosis of CM.