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1.
J Eukaryot Microbiol ; 70(4): e12975, 2023.
Article in English | MEDLINE | ID: mdl-36972021

ABSTRACT

The health of coastal marine environments is severely declining with global changes. Proxies, such as those based on microeukaryote communities, can record biodiversity and ecosystem responses. However, conventional studies rely on microscopic observations of limited taxonomic range and size fraction, missing putatively ecologically informative community components. Here, we tested molecular tools to survey foraminiferal biodiversity in a fjord system (Sweden) on spatial and temporal scales: Alpha and beta diversity responses to natural and anthropogenic environmental trends were assessed and variability of foraminiferal environmental DNA (eDNA) compared to morphology-based data. The identification of eDNA-obtained taxonomic units was aided by single-cell barcoding. Our study revealed wide diversity, including typical morphospecies recognized in the fjords, and so-far unrecognized taxa. DNA extraction method impacted community composition outputs significantly. DNA extractions of 10 g sediment more reliably represented present diversity than of 0.5-g samples and, thus, are preferred for environmental assessments in this region. Alpha- and beta diversity of 10-g extracts correlated with bottom-water salinity similar to morpho-assemblage diversity changes. Sub-annual environmental variability resolved only partially, indicating damped sensitivity of foraminiferal communities on short timescales using established metabarcoding techniques. Systematically addressing the current limitations of morphology-based and metabarcoding studies may strongly improve future biodiversity and environmental assessments.


Subject(s)
DNA, Environmental , Foraminifera , Ecosystem , Estuaries , DNA, Environmental/genetics , Foraminifera/genetics , Environmental Monitoring/methods , Biodiversity , DNA , DNA Barcoding, Taxonomic
2.
PLoS One ; 10(8): e0135579, 2015.
Article in English | MEDLINE | ID: mdl-26275216

ABSTRACT

Staphylococcus aureus is an important pathogen manifesting virulence through diverse disease forms, ranging from acute skin infections to life-threatening bacteremia or systemic toxic shock syndromes. In the latter case, the prototypical superantigen is TSST-1 (Toxic Shock Syndrome Toxin 1), encoded by tst(H), and carried on a mobile genetic element that is not present in all S. aureus strains. Transcriptional regulation of tst is only partially understood. In this study, we dissected the role of sarA, sarS (sarH1), RNAIII, rot, and the alternative stress sigma factor sigB (σB). By examining tst promoter regulation predominantly in the context of its native sequence within the SaPI1 pathogenicity island of strain RN4282, we discovered that σB emerged as a particularly important tst regulator. We did not detect a consensus σB site within the tst promoter, and thus the effect of σB is likely indirect. We found that σB strongly repressed the expression of the toxin via at least two distinct regulatory pathways dependent upon sarA and agr. Furthermore rot, a member of SarA family, was shown to repress tst expression when overexpressed, although its deletion had no consistent measurable effect. We could not find any detectable effect of sarS, either by deletion or overexpression, suggesting that this regulator plays a minimal role in TSST-1 expression except when combined with disruption of sarA. Collectively, our results extend our understanding of complex multifactorial regulation of tst, revealing several layers of negative regulation. In addition to environmental stimuli thought to impact TSST-1 production, these findings support a model whereby sporadic mutation in a few key negative regulators can profoundly affect and enhance TSST-1 expression.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , DNA-Binding Proteins/metabolism , Enterotoxins/metabolism , Promoter Regions, Genetic/genetics , Repressor Proteins/metabolism , Sigma Factor/metabolism , Staphylococcus aureus/metabolism , Superantigens/metabolism , Trans-Activators/metabolism , Bacterial Proteins/genetics , Bacterial Toxins/genetics , DNA-Binding Proteins/genetics , Enterotoxins/genetics , Gene Expression Regulation, Bacterial/genetics , Genomic Islands/genetics , Repressor Proteins/genetics , Sigma Factor/genetics , Superantigens/genetics , Trans-Activators/genetics
3.
Antimicrob Agents Chemother ; 59(4): 1922-30, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25583724

ABSTRACT

The development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested the in vitro activity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 µg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 µg/ml; resistant [R], >1 µg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Cephalosporins/pharmacology , Cross Infection/microbiology , Methicillin-Resistant Staphylococcus aureus/drug effects , Mutation, Missense/genetics , Staphylococcal Infections/microbiology , Drug Resistance, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Penicillin-Binding Proteins , Switzerland/epidemiology , Ceftaroline
4.
PLoS One ; 8(9): e77122, 2013.
Article in English | MEDLINE | ID: mdl-24098817

ABSTRACT

Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.


Subject(s)
Genetic Variation/genetics , Genome, Bacterial/genetics , Staphylococcus aureus/genetics , Amino Acid Sequence , Base Sequence , Computational Biology , High-Throughput Nucleotide Sequencing/methods , INDEL Mutation/genetics , Molecular Sequence Data , Pedigree , Polymorphism, Single Nucleotide/genetics , Real-Time Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Species Specificity
5.
Antimicrob Agents Chemother ; 57(7): 3283-92, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23629700

ABSTRACT

S. aureus combats cell wall antibiotic stress by altered gene expression mediated by various environmental signal sensors. In this study, we examined the transcriptional regulation of trfA, a gene related to mecA of Bacillus subtilis encoding an adaptor protein implicated in multiple roles, notably, proteolysis and genetic competence. Despite strong sequence similarity to B. subtilis mecA, the function of S. aureus trfA remains largely unexplored; however, its deletion leads to almost complete loss of resistance to oxacillin and glycopeptide antibiotics in glycopeptide-intermediate S. aureus (GISA) derivatives of methicillin-susceptible or methicillin-resistant S. aureus (MRSA) clinical or laboratory isolates. Northern blot analysis and 5' rapid amplification of cDNA ends (RACE) mapping revealed that trfA was expressed monocistronically by three promoters. Cell wall-active antibiotic exposure led to both increased trfA transcription and enhanced steady-state TrfA levels. trfA promoter regulation was not dependent upon the cell wall stress sentinel VraSR and other sensory stress systems, such as GraRS, WalkRK, Stk1/Stp1, and SigB. Notably, we discovered that the global oxidative-stress regulator Spx controlled trfA transcription. This finding was also confirmed using a strain with enhanced Spx levels resulting from a defect in yjbH, encoding a Spx-interacting protein governing Spx proteolytic degradation. A cohort of clinical GISA strains revealed significant steady-state upregulation of trfA compared to corresponding susceptible parental strains, further supporting a role for trfA in antibiotic resistance. These data provide strong evidence for a link between cell wall antibiotic stress and evoked responses mediated by an oxidative-stress sensor.


Subject(s)
Bacterial Proteins/genetics , Cell Wall/genetics , Drug Resistance, Multiple, Bacterial/genetics , Gene Expression Regulation, Bacterial , Membrane Proteins/genetics , Staphylococcus aureus/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Base Sequence , Cell Wall/drug effects , Methicillin Resistance/genetics , Molecular Sequence Data , Oxacillin/pharmacology , Oxidative Stress/genetics , Promoter Regions, Genetic , Sequence Analysis, DNA , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purification , Sulfhydryl Compounds/metabolism , Transcription, Genetic
6.
PLoS One ; 6(6): e21577, 2011.
Article in English | MEDLINE | ID: mdl-21738716

ABSTRACT

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Microbial/genetics , Genome, Bacterial/genetics , Glycopeptides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Microbial Sensitivity Tests
7.
Antimicrob Agents Chemother ; 55(3): 1008-20, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21173175

ABSTRACT

An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Glycopeptides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Bacterial Proteins/genetics , Blotting, Northern , DNA-Binding Proteins/genetics , Microbial Sensitivity Tests , Mutation , Polymerase Chain Reaction , Staphylococcus aureus/genetics
8.
Antimicrob Agents Chemother ; 53(3): 903-11, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19104009

ABSTRACT

Endogenous, low-level glycopeptide resistance in Staphylococcus aureus results from multifactorial genetic changes. Comparative genomic hybridization analysis revealed the specific deletion of a 1.8-kb segment encompassing two adjacent open reading frames (ORFs) of unknown function in a teicoplanin-susceptible revertant (strain 14-4rev) compared to the sequence of its isogenic, teicoplanin-resistant parental strain, strain 14-4. This provocative finding prompted us to perform a detailed genetic analysis of the contribution of this genomic segment to glycopeptide resistance. Despite repeated efforts in our laboratory, 14-4 and 14-4rev have proven refractory to most genetic manipulations. To circumvent this difficulty, we evaluated the contribution of both putative ORFs (designated teicoplanin resistance factors trfA and trfB) on teicoplanin resistance in a different, genetically tractable background. Genetic analysis showed that single or double trfA and/or trfB mutations abolished teicoplanin resistance in two independent teicoplanin-resistant derivatives of NCTC8325 strain ISP794 generated by two-step passages with the drug. The frequency of teicoplanin-resistant mutants was markedly decreased by the absence of trfAB in the teicoplanin-susceptible ISP794 background. Nevertheless, a low rate of teicoplanin-resistant mutants was selected from ISP794 trfAB, thus indicating an additional contribution of trfAB-independent pathways in the emergence of low-level glycopeptide resistance. Further experiments performed with clinical glycopeptide-intermediate S. aureus isolate NRS3 indicated that the trfAB mutation could affect not only teicoplanin resistance but also vancomycin and oxacillin resistance. In conclusion, our study demonstrates the key role of two novel loci in endogenous, low-level glycopeptide resistance in S. aureus whose precise molecular functions warrant further investigation.


Subject(s)
Anti-Bacterial Agents/pharmacology , Genome, Bacterial/drug effects , Glycopeptides/genetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Comparative Genomic Hybridization , Drug Resistance, Microbial , Glycopeptides/metabolism , Humans , Microbial Sensitivity Tests , Mutation/drug effects , Staphylococcus aureus/metabolism , Teicoplanin/pharmacology , Vancomycin Resistance/drug effects
9.
Antimicrob Agents Chemother ; 50(9): 3048-61, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16940101

ABSTRACT

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.


Subject(s)
Methicillin Resistance , Staphylococcus aureus/physiology , Teicoplanin/pharmacology , Bacteriolysis/genetics , Cell Wall/genetics , Extracellular Fluid/enzymology , Humans , Hydrolases/metabolism , Octoxynol , Oligonucleotide Array Sequence Analysis , Peptide Hydrolases/biosynthesis , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Transcription, Genetic
10.
J Microbiol Methods ; 61(2): 201-8, 2005 May.
Article in English | MEDLINE | ID: mdl-15722146

ABSTRACT

Rapid and accurate identification and speciation of staphylococci clinical isolates is important for predicting medical pathology. We evaluated the ability of a high-density DNA probe array based on 16S rDNA sequences to identify Staphylococcus species. Correct identification was observed for 185 out of the 201 strains (92%). Of the 33 tested species, the array was able to correctly identify 30 of them. The total time required for identification of 4 isolates was 5 h. Such a tool represents a powerful method for routine microbiological diagnostic as well as for epidemiological studies.


Subject(s)
DNA Probes/genetics , Oligonucleotide Array Sequence Analysis/methods , Staphylococcus/classification , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA Probes/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Polymerase Chain Reaction/methods , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Staphylococcus/genetics , Staphylococcus/isolation & purification , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics
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