Genetic characterization of staphopain genes in Staphylococcus aureus

2004 ◽  
Vol 385 (11) ◽  
pp. 1059-1067 ◽  
Author(s):  
Ewa Golonka ◽  
Renata Filipek ◽  
Artur Sabat ◽  
Anna Sinczak ◽  
Jan Potempa
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Proteolytic Enzymes ◽  
Disease Process ◽  
Pcr Amplification ◽  
Single Copy ◽  
Pcr Rflp ◽  
Genes Encoding

AbstractStaphylococcus aureus, a leading cause of bacterial infections in humans, is endowed with a wealth of virulence factors that contribute to the disease process. Several extracellular proteolytic enzymes, including cysteine proteinases referred to as the staphopains (staphopain A, encoded by thescpAgene, and staphopain B, encoded bysspB), have proposed roles for staphylococcal virulence. Here we present data regarding the distribution, copy number and genetic variability of the genes encoding the staphopains in a large number ofS. aureusstrains. The polymorphism of thescpAandsspBgenes in three laboratory strains and 126 clinical isolates was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Both genes were detected in all isolates by PCR amplification and, based on the PCR-RFLP patterns, classified as four types forscpAand six types forsspB. Those with the most divergent patterns were subjected to DNA sequencing and compared with genomic sequence data for the seven available strains ofS. aureus. Southern blot analysis of thescpAandsspBsequences indicates that they are strongly conserved as single-copy genes in the genome of eachS. aureusstrain investigated. Taken together, these data suggest that the staphopains have important housekeeping and/or virulence functions, and therefore may constitute an interesting target for the development of therapeutic inhibitors for the treatment of staphylococcal diseases.

scholarly journals In VitroStudies Indicate a High Resistance Potential for the Lantibiotic Nisin in Staphylococcus aureus and Define a Genetic Basis for Nisin Resistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2362-2368 ◽  
Author(s):  
Katy L. Blake ◽  
Chris P. Randall ◽  
Alex J. O'Neill
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Fold Increase ◽  
Peptide Antibiotics ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Development Of Resistance ◽  
Genes Encoding ◽  
Bacterial Fitness

ABSTRACTLantibiotics such as nisin (NIS) are peptide antibiotics that may have a role in the chemotherapy of bacterial infections. A perceived benefit of lantibiotics for clinical use is their low propensity to select resistance, although detailed resistance studies with relevant bacterial pathogens are lacking. Here we examined the development of resistance to NIS inStaphylococcus aureus, establishing that mutants, including small-colony variants, exhibiting substantial (4- to 32-fold) reductions in NIS susceptibility could be selected readily. Comparative genome sequencing of a single NISrmutant exhibiting a 32-fold increase in NIS MIC revealed the presence of only two mutations, leading to the substitutions V229G in the purine operon repressor, PurR, and A208E in an uncharacterized protein encoded by SAOUHSC_02955. Independently selected NISrmutants also harbored mutations in the genes encoding these products. Reintroduction of these mutations into theS. aureuschromosome alone and in combination revealed that SAOUHSC_02955(A208E) made the primary contribution to the resistance phenotype, conferring up to a 16-fold decrease in NIS susceptibility. Bioinformatic analyses suggested that this gene encodes a sensor histidine kinase, leading us to designate it “nisin susceptibility-associated sensor (nsaS).” Doubling-time determinations and mixed-culture competition assays between NISrand NISsstrains indicated that NIS resistance had little impact on bacterial fitness, and resistance was stable in the absence of selection. The apparent ease with whichS. aureuscan develop and maintain NIS resistancein vitrosuggests that resistance to NIS and other lantibiotics with similar modes of action would arise in the clinic if these agents are employed as chemotherapeutic drugs.

scholarly journals Measuring genome sizes using read-depth, k-mers, and flow cytometry: methodological comparisons in beetles (Coleoptera)

2019 ◽  
Author(s):  
James M. Pflug ◽  
Valerie Renee Holmes ◽  
Crystal Burrus ◽  
J. Spencer Johnston ◽  
David R. Maddison
Keyword(s):  
Flow Cytometry ◽  
Genome Size ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Read Depth ◽  
Test System ◽  
Estimation Methods ◽  
Next Generation

ABSTRACTMeasuring genome size across different species can yield important insights into evolution of the genome and allow for more informed decisions when designing next-generation genomic sequencing projects. New techniques for estimating genome size using shallow genomic sequence data have emerged which have the potential to augment our knowledge of genome sizes, yet these methods have only been used in a limited number of empirical studies. In this project, we compare estimation methods using next-generation sequencing (k-mer methods and average read depth of single-copy genes) to measurements from flow cytometry, the gold standard for genome size measures, using ground beetles (Carabidae) and other members of the beetle suborder Adephaga as our test system. We also present a new protocol for using read-depth of single-copy genes to estimate genome size. Additionally, we report flow cytometry measurements for five previously unmeasured carabid species, as well as 21 new draft genomes and six new draft transcriptomes across eight species of adephagan beetles. No single sequence-based method performed well on all species, and all tended to underestimate the genome sizes, although only slightly in most samples. For one species, Bembidion haplogonum, most sequence-based methods yielded estimates half the size suggested by flow cytometry. This discrepancy for k-mer methods can be explained by a large number of repetitive sequences, but we have no explanation for why read-depth methods yielded results that were also strikingly low.

Defects in pyruvate kinase cause a conditional increase of thiamine synthesis inSalmonella typhimurium

1999 ◽  
Vol 45 (7) ◽  
pp. 565-572 ◽  
Author(s):  
Todd Christian ◽  
Diana M Downs
Keyword(s):  
Pyruvate Kinase ◽  
De Novo ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Purine Biosynthesis ◽  
Phenotypic Analysis ◽  
Metabolic Defects ◽  
Genes Encoding ◽  
Metabolic Interactions

As genomic sequence data become more prevalent, the challenges in microbial physiology shift from identifying biochemical pathways to understanding the interactions that occur between them to create a robust but responsive metabolism. One of the most powerful methods to identify such interactions is in vivo phenotypic analysis. We have utilized thiamine synthesis as a model to detect subtle metabolic interactions due to the sensitivity allowed by the small cellular requirement for this vitamin. Although purine biosynthesis produces an intermediate in thiamine synthesis, mutants blocked in the first step of de novo purine biosynthesis (PurF) are able to grow in the absence of thiamine owing to an alternative synthesis. A number of general metabolic defects have been found to prevent PurF-independent thiamine synthesis. Here we report stimulation of thiamine-independent growth caused by a mutation in one or both genes encoding the pyruvate kinase isozymes. The results presented herein represent the first phenotype described for mutants defective in pykA or pykF, and thus identify metabolic interactions that exist in vivo.Key words: thiamine synthesis, metabolic integration.

scholarly journals Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Allison Black ◽  
Louise H. Moncla ◽  
Katherine Laiton-Donato ◽  
Barney Potter ◽  
Lissethe Pardo ◽  
...  
Keyword(s):  
Zika Virus ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Epidemiological Studies ◽  
Multiple Introductions ◽  
Genomic Epidemiology ◽  
Definition Of ◽  
And Control ◽  
Affected Country

Abstract Background Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. Methods We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. Results We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 – Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. Conclusions Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia.

scholarly journals Atypical Presentation of Methicillin-Susceptible Staphylococcus aureus Infection in a Dengue-Positive Patient: A Case Report with Virulence Genes Analysis

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 190
Author(s):  
Soo Tein Ngoi ◽  
Yee Wan Lee ◽  
Wen Kiong Niek ◽  
Foong Kee Kan ◽  
Sazaly AbuBakar ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dengue Fever ◽  
Genomic Sequence ◽  
Protein A ◽  
Mortality And Morbidity ◽  
Multiple Organs ◽  
Patient Reported ◽  
Mssa Strain ◽  
Genes Encoding ◽  
Staphylococcal Pneumonia

Concurrent bacteraemia in patients with dengue fever is rarely reported. We report a case of a patient who initially presented with symptoms typical of dengue fever but later succumbed to septic shock caused by hypervirulent methicillin-susceptible Staphylococcus aureus (MSSA). A 50-year-old female patient with hypertension and diabetes mellitus presented with typical symptoms of dengue fever. Upon investigation, the patient reported having prolonged fever for four days prior to hospitalization. Within 24 hours post-admission, the patient developed pneumonia and refractory shock, and ultimately succumbed to multiple-organs failure. Microbiological examination of the blood culture retrieved a pan susceptible MSSA strain. Genomic sequence analyses of the MSSA strain identified genes encoding staphylococcal superantigens (enterotoxin staphylococcal enterotoxin C 3 (SEC3) and enterotoxin-like staphylococcal enterotoxins-like toxin L (SElL)) that have been associated with toxic shock syndrome in human hosts. Genes encoding important toxins (Panton-Valentine leukocidins, alpha-haemolysin, protein A) involved in the development of staphylococcal pneumonia were also present in the MSSA genome. Staphylococcus aureus co-infections in dengue are uncommon but could be exceptionally fatal if caused by a toxin-producing strain. Clinicians should be aware of the risks and signs of sepsis in dengue fever, thus allowing early diagnosis and starting of antibiotic treatment in time to lower the mortality and morbidity rates.

scholarly journals PCR-RFLP: a targeted method to reveal host specific malacosporean infection profiles (Cnidaria: Myxozoa: Malacosporea)

2020 ◽  
Vol 141 ◽  
pp. 91-101
Author(s):  
P Ruggeri ◽  
J Naldoni ◽  
H Hartikainen ◽  
B Okamura
Keyword(s):  
Sanger Sequencing ◽  
Sequence Data ◽  
Pcr Amplification ◽  
18S Rrna Gene ◽  
Single Step ◽  
Rrna Gene ◽  
Mixed Infections ◽  
Intermediate Hosts ◽  
Tissue Samples ◽  
Pcr Rflp

Malacosporeans are a group of endoparasitic cnidarians (Myxozoa) that use freshwater bryozoans and fish as final and intermediate hosts, respectively. The malacosporean Tetracapsuloides bryosalmonae causes proliferative kidney disease (PKD), an emerging disease in aquaculture and wild fish populations, including threatened salmonids in Europe and the USA. Mixed infections of malacosporeans are often encountered, and a monitoring tool for screening of multiple malacosporean species in either their fish or bryozoan hosts is therefore desirable. We describe an inexpensive method that combines PCR amplification of the partial 18S rRNA gene (~260 bp) and a single-step restriction fragment length polymorphism (RFLP) method for identification of 10 malacosporean lineages and species. We demonstrate and test this methodology on a set of DNA extracted from malacosporeans infecting fish kidney and tissues sampled from bryozoan colonies and compare the results with Sanger sequencing of the same parasite DNA isolates. The PCR-RFLP and Sanger sequencing methods agreed in 100% of cases. The PCR-RFLP method offers a number of opportunities, including screening large panels of host tissue samples to gain insights into infection patterns, characterizing mixed infections, and confirming highly pathogenic T. bryosalmonae infections. The method can also be further refined as new sequence data become available for malacosporeans.

scholarly journals Measuring Genome Sizes Using Read-Depth, k-mers, and Flow Cytometry: Methodological Comparisons in Beetles (Coleoptera)

2020 ◽  
Vol 10 (9) ◽  
pp. 3047-3060 ◽  
Author(s):  
James M. Pflug ◽  
Valerie Renee Holmes ◽  
Crystal Burrus ◽  
J. Spencer Johnston ◽  
David R. Maddison
Keyword(s):  
Flow Cytometry ◽  
Genome Size ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Empirical Studies ◽  
Single Copy ◽  
Read Depth ◽  
Test System ◽  
Estimation Methods ◽  
Next Generation

Measuring genome size across different species can yield important insights into evolution of the genome and allow for more informed decisions when designing next-generation genomic sequencing projects. New techniques for estimating genome size using shallow genomic sequence data have emerged which have the potential to augment our knowledge of genome sizes, yet these methods have only been used in a limited number of empirical studies. In this project, we compare estimation methods using next-generation sequencing (k-mer methods and average read depth of single-copy genes) to measurements from flow cytometry, a standard method for genome size measures, using ground beetles (Carabidae) and other members of the beetle suborder Adephaga as our test system. We also present a new protocol for using read-depth of single-copy genes to estimate genome size. Additionally, we report flow cytometry measurements for five previously unmeasured carabid species, as well as 21 new draft genomes and six new draft transcriptomes across eight species of adephagan beetles. No single sequence-based method performed well on all species, and all tended to underestimate the genome sizes, although only slightly in most samples. For one species, Bembidion sp. nr. transversale, most sequence-based methods yielded estimates half the size suggested by flow cytometry.

scholarly journals Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia

2018 ◽  
Author(s):  
Allison Black ◽  
Louise H. Moncla ◽  
Katherine Laiton-Donato ◽  
Barney Potter ◽  
Lissethe Pardo ◽  
...  
Keyword(s):  
Zika Virus ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Epidemiologic Studies ◽  
Multiple Introductions ◽  
Genomic Epidemiology ◽  
Definition Of ◽  
And Control ◽  
Affected Country

AbstractBackgroundColombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia.MethodsWe sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes ae-gypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia.ResultsWe attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeo-graphic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela.ConclusionsSimilarly to genomic epidemiologic studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. This more accurate dating of when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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