scholarly journals Optimized genotyping method for identification of bacterial contaminants in pharmaceutical industry

2016 ◽  
Vol 66 (2) ◽  
pp. 289-295
Author(s):  
Borche Stamatoski ◽  
Miroslava Ilievska ◽  
Hristina Babunovska ◽  
Nikola Sekulovski ◽  
Sasho Panov
Keyword(s):  
Pharmaceutical Industry ◽  
Dna Sequencing ◽  
Bacterial Contamination ◽  
Raw Materials ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Bacterial Contaminants ◽  
Microbiological Control ◽  
Selection Of

AbstractMicrobiological control is of crucial importance in the pharmaceutical industry regarding the possible bacterial contamination of the environment, water, raw materials and finished products. Molecular identification of bacterial contaminants based on DNA sequencing of the hypervariable 16SrRNA gene has been introduced recently. The aim of this study is to investigate the suitability of gene sequencing using our selection of PCR primers and conditions for rapid and accurate bacterial identification in pharmaceutical industry quality control.DNA was extracted from overnight incubated colonies from 10 bacterial ATCC strains, which are common contaminants in the pharmaceutical industry. A region of bacterial 16SrRNA gene was analyzed by bidirectional DNA sequencing. Bacterial identification based on partial sequencing of the 16SrRNA gene is the appropriate method that could be used in the pharmaceutical industry after adequate validations. We have successfully identified all tested bacteria with more than 99 % similarity to the already published sequences.

scholarly journals PCR-Based Detection of Angiostrongylus cantonensis in Tissue and Mucus Secretions from Molluscan Hosts

2006 ◽  
Vol 73 (5) ◽  
pp. 1415-1419 ◽  
Author(s):  
Yvonne Qvarnstrom ◽  
James J. Sullivan ◽  
Henry S. Bishop ◽  
Robert Hollingsworth ◽  
Alexandre J. da Silva
Keyword(s):  
Dna Sequencing ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Angiostrongylus Cantonensis ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Sequencing Method ◽  
Third Stage ◽  
Dna Sequencing Analysis

ABSTRACT Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Rapid, Specific, and Sensitive Detection of Spoilage Molds in Orange Juice Using a Real-Time Taqman PCR Assay

2006 ◽  
Vol 69 (2) ◽  
pp. 385-390 ◽  
Author(s):  
KAI WAN ◽  
AHMED E. YOUSEF ◽  
STEVE J. SCHWARTZ ◽  
HUA H. WANG
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Orange Juice ◽  
Raw Materials ◽  
Pcr Primers ◽  
Cross Reactivity ◽  
Rrna Gene ◽  
Control Analysis ◽  
Financial Loss ◽  
Food Ingredients

The outgrowth of spoilage organisms, including molds and yeasts, results in significant financial loss to the food industry and wastes natural resources. The objective of this study was to develop a rapid, specific, and sensitive real-time PCR method for detecting spoilage molds during screening of raw materials and final product quality control analysis. The 18S rRNA gene was used to develop PCR primers and probe. With this set of primers and probe, less than 1,000 mold cells per milliliter of orange juice (10 cells per reaction) were detected with the real-time PCR system within 6 to 7 h. No cross-reactivity was found with other common foodborne bacteria, yeasts, or food ingredients. This technique is significantly faster than current detection and identification procedures, which take from days to weeks.

scholarly journals Bacterial Contamination of Boar Semen and its Relationship to Sperm Quality Preserved in Commercial Extender Containing Gentamicin Sulfate

2016 ◽  
Vol 19 (3) ◽  
pp. 451-459 ◽  
Author(s):  
D. Gączarzewicz ◽  
J. Udała ◽  
M. Piasecka ◽  
B. Błaszczyk ◽  
T. Stankiewicz
Keyword(s):  
Bacterial Contamination ◽  
Antimicrobial Agents ◽  
Sperm Quality ◽  
Aerobic Bacteria ◽  
Large White ◽  
Gentamicin Sulfate ◽  
Bacterial Contaminants ◽  
Microbiological Control ◽  
Boar Semen ◽  
High Standards

Abstract This study was designed to determine the degree and type of bacterial contamination in boar semen (79 ejaculates from Large White and Landrace boars) and its consequences for sperm quality during storage (27 extended semen samples, 16°C for five days) under practical conditions of artificial insemination (AI). The results revealed the presence of aerobic bacteria in 99% of the ejaculates (from 80 to 370 ×106 colony-forming units/mL). Most of the ejaculates contained two or three bacterial contaminants, while the Staphylococcus, Streptococcus, and Pseudomonas bacterial genera were most frequently isolated. Also detected were Enterobacter spp., Bacillus spp., Proteus spp., Escherichia coli, P. fluorescens, and P. aeruginosa. In general, the growth of certain bacterial types isolated prior to semen processing (Enterobacter spp., E. coli, P. fluorescens, and P. aeruginosa) was not discovered on different days of storage, but fluctuations (with a tendency towards increases) were found in the frequencies of Bacillus spp., Pseudomonas spp., and Staphylococcus spp. isolates up to the end of storage. Semen preserved for five days exhibited decreases in sperm motility and increases in the average number of total aerobic bacteria; this was associated with sperm agglutination, plasma membrane disruption, and acrosome damage. We inferred that, due to the different degrees and types of bacterial contaminants in the boar ejaculates, the inhibitory activity of some antimicrobial agents used in swine extenders (such as gentamicin sulfate) may be limited. Because such agents can contribute to the overgrowth of certain aerobic bacteria and a reduction in the quality of stored semen, procedures with high standards of hygiene and microbiological control should be used when processing boar semen.

BÖHLER W403 VMR

Alloy Digest ◽  
2013 ◽  
Vol 62 (9) ◽  
Keyword(s):  
Heat Treatment ◽  
Chemical Composition ◽  
Physical Properties ◽  
Tool Steel ◽  
Raw Materials ◽  
Heat Treating ◽  
Diffusion Annealing ◽  
Selection Of

Abstract Böhler (or Boehler) W403 VMR is a tool steel with outstanding properties, based not only on a modified chemical composition, but on the selection of highly clean raw materials for melting, remelting under vacuum (VMF), optimized diffusion annealing, and a special heat treatment. This datasheet provides information on composition, physical properties, and elasticity. It also includes information on forming and heat treating. Filing Code: TS-721. Producer or source: Böhler Edelstahl GmbH.

scholarly journals Isolation and Characterization of Lactic Acid Bacteria and Yeasts from Typical Bulgarian Sourdoughs

2021 ◽  
Vol 9 (7) ◽  
pp. 1346
Author(s):  
Mariana Petkova ◽  
Petya Stefanova ◽  
Velitchka Gotcheva ◽  
Angel Angelov
Keyword(s):  
Lactic Acid ◽  
Sequence Analysis ◽  
Lactic Acid Bacteria ◽  
Antimicrobial Properties ◽  
Matter Content ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Dry Matter Content ◽  
Isolation And Characterization ◽  
Selection Of

Traditional sourdoughs in Bulgaria were almost extinct during the centralized food production system. However, a rapidly developing trend of sourdough revival in the country is setting the demand for increased production and use of commercial starter cultures. The selection of strains for such cultures is based on geographical specificity and beneficial technological properties. In this connection, the aim of this study was to isolate, identify and characterize lactic acid bacteria (LAB) and yeasts from typical Bulgarian sourdoughs for the selection of strains for commercial sourdough starter cultures. Twelve samples of typical Bulgarian sourdoughs were collected from different geographical locations. All samples were analyzed for pH, total titratable acidity and dry matter content. Enumeration of LAB and yeast was also carried out. Molecular identification by 16S rDNA sequence analysis was performed for 167 LAB isolates, and 106 yeast strains were identified by ITS1-5.8S-ITS2 rRNA gene partial sequence analysis. The LAB strains were characterized according to their amylolytic and proteolytic activity and acidification capacity, and 11 strains were selected for further testing of their antimicrobial properties. The strains with the most pronounced antibacterial and antifungal activity are listed as recommended candidates for the development of starter cultures for sourdoughs or other food products.

scholarly journals Citrobacter tructae sp. nov. Isolated from Kidney of Diseased Rainbow Trout (Oncorhynchus mykiss)

2021 ◽  
Vol 9 (2) ◽  
pp. 275
Author(s):  
Won Joon Jung ◽  
Hyoun Joong Kim ◽  
Sib Sankar Giri ◽  
Sang Guen Kim ◽  
Sang Wha Kim ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Biochemical Characterization ◽  
Antibiotic Resistance Genes ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
The 16S Rrna Gene ◽  
Genome Distance

A novel Citrobacter species was isolated from the kidney of diseased rainbow trout (Oncorhynchus mykiss) reared on a trout farm. Biochemical characterization and phylogenetic analysis were performed for bacterial identification. Sequencing of the 16S rRNA gene and five housekeeping genes indicated that the strain belongs to the Citrobacter genus. However, multilocus sequence analysis, a comparison of average nucleotide identity, and genome-to-genome distance values revealed that strain SNU WT2 is distinct and forms a separate clade from other Citrobacter species. Additionally, the phenotype characteristics of the strain differed from those of other Citrobacter species. Quinone analysis indicated that the predominant isoprenoid quinone is Q-10. Furthermore, strain virulence was determined by a rainbow trout challenge trial, and the strain showed resistance to diverse antibiotics including β-lactams, quinolone, and aminoglycosides. The complete genome of strain SNU WT2 is 4,840,504 bp with a DNA G + C content of 51.94% and 106,068-bp plasmid. Genome analysis revealed that the strain carries virulence factors on its chromosome and antibiotic resistance genes on its plasmid. This strain represents a novel species in the genus Citrobacter for which the name C. tructae has been proposed, with SNU WT2 (=KCTC 72517 = JCM 33612) as the type strain.

scholarly journals Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Otun Saha ◽  
Nadira Naznin Rakhi ◽  
Arif Istiaq ◽  
Israt Islam ◽  
Munawar Sultana ◽  
...  
Keyword(s):  
Micrococcus Luteus ◽  
Bacterial Species ◽  
16S Rrna Gene Sequencing ◽  
Average Diameter ◽  
Diffusion Method ◽  
Dilution Method ◽  
Rrna Gene ◽  
Poultry Farms ◽  
Rrna Gene Sequencing ◽  
Selection Of

Introduction. Effective sanitation strategies for poultry farms require an appropriate selection of the disinfectant based on the contaminants present and their sensitivity to the disinfectants. Aim. The current study investigated the prevalence of streptococci/micrococci in poultry farms of Bangladesh and the efficacy of commercial disinfectants (Savlon, Lysol, Quatovet, Virkon S, and Virocid) along with alcohol against these pathogens to adopt appropriate strategies. Materials and Methods. Conventional approaches and the 16S rRNA gene sequencing were performed to confirm the isolates at the species level along with microtiter biofilm assay to determine their biofilm-forming ability. Efficacy of the disinfectants was tested against those isolates using agar well diffusion and minimum inhibitory concentration (MIC) test by broth dilution method using different dilutions of the disinfectants. Results. Staphylococcus lentus (n = 32), Micrococcus luteus (n = 7), and Micrococcus aloeverae (n = 4) were confirmed among 102 presumptively screened streptococci/micrococci isolates from 43 samples. No single disinfectant showed equally high efficacy against all three bacterial species in agar well diffusion test, although Virocid showed the lowest MIC against all three of them. Lysol was least effective among the commercial disinfectants by both MIC and diffusion method, although each commercial disinfectant was more effective than alcohol. Considering both the average diameter of the inhibition zones and the MIC values, efficacy can be interpreted as Virocid > Quatovet > Savlon > Virkon S > Lysol. Although the efficacy decreased with decreasing concentration, the disinfectants retained a satisfactory level of efficacy at 50% concentration. Among test pathogens, M. aloeverae was the most sensitive to the disinfectants and the weakest biofilm producers, whereas 4/14 S. lentus and 1/5 M. luteus were strong biofilm producers, which may cause more reduction in the efficacy in environmental conditions. Conclusion. As no ideal disinfectant was found in the study, the efficacy of the disinfectants should be routinely evaluated and validated to ensure the sanitation standards in the poultry sector.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

Sign in / Sign up

Export Citation Format

Share Document