Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus

2021 ◽  
Vol 70 (6) ◽  
Author(s):  
Elyse C. Curry ◽  
Ryan G. Hart ◽  
Danni Y. Habtu ◽  
Neal R. Chamberlain
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Type Species ◽  
Proteinase K ◽  
Partial Characterization ◽  
Content Type ◽  
Link Type ◽  
Inducing Factors

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci. Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci. Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000. Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications. Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis ’ PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus ’ PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis . PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells. Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.

scholarly journals 1194. Identification and Characterization of Extracellular Inducers of Persistence in Staphylococcus epidermidis and Staphylococcus aureus

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S619-S619
Author(s):  
Danni Y Habtu ◽  
Neal Chamberlain ◽  
Elyse Curry ◽  
Ryan Hart
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Proteinase K ◽  
Bacterial Cells ◽  
Staphylococcal Species ◽  
Inducing Factors ◽  
Proteinase K Treatment ◽  
Human Infections

Abstract Background This study describes the identification and partial characterization of persistence inducing factors (PIF) from Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Persistence is an epigenetic process that results in tolerance of bacterial cells to antibiotic treatment, which can result in chronic human infections. Methods Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCF) from S. epidermidis RP62A and S. aureus SH1000 were obtained at various OD600’s and following incubation at 16 h. The CCF’s were used to develop a persistence inducing factor (PIF) assay. The PIF assay was used to partially characterize PIF from S. epidermidis RP62A and S. aureus SH1000 for relative molecular weight, temperature and protease sensitivity and inter-species communications. Results Optimal OD600’s for the S. epidermidis RP62A and S. aureus SH1000 PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis RP62A’s PIF activity was decreased by storage at 4o C (2 weeks or longer) but not following incubation at 20o C (16 h), 37o C (1 h) or 100o C (15 min). S. aureus SH1000’s PIF activity was decreased following storage at 4o C (2 week or longer) and after boiling at 100oC for 5 min but not after incubation at 37o C (1 h). PIF activity from both species was less than 3,000 Mrr. Proteinase-K treatment of S. aureus SH1000 PIF decreased activity but did not decrease PIF activity of S. epidermidis RP62A. PIF from S. epidermidis RP62A did not increase persister numbers when used to treat S. aureus SH1000 cells nor did PIF from S. aureus SH1000 increase persister numbers in S. epidermidis RP62A cells. Conclusion Previous attempts to discover PIF’s for staphylococcal species were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species appear to produce unique, extracellular, low-molecular-weight inducers of persistence (PIF) when assayed using an OD600-based PIF assay. Disclosures All Authors: No reported disclosures

Impact of pH on growth of Staphylococcus epidermidis and Staphylococcus aureus in vitro

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Vidula Iyer ◽  
Janhavi Raut ◽  
Anindya Dasgupta
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Kinetics ◽  
Staphylococcus Epidermidis ◽  
Type Species ◽  
Ph Dependence ◽  
Skin Microbiome ◽  
Content Type ◽  
Link Type ◽  
Kinetics Of

The pH of skin is critical for skin health and resilience and plays a key role in controlling the skin microbiome. It has been well reported that under dysbiotic conditions such as atopic dermatitis (AD), eczema, etc. there are significant aberrations of skin pH, along with a higher level of Staphylococcus aureus compared to the commensal Staphylococcus epidermidis on skin. To understand the effect of pH on the relative growth of S. epidermidis and S. aureus , we carried out simple in vitro growth kinetic studies of the individual microbes under varying pH conditions. We demonstrated that the growth kinetics of S. epidermidis is relatively insensitive to pH within the range of 5–7, while S. aureus shows a stronger pH dependence in that range. Gompertz’s model was used to fit the pH dependence of the growth kinetics of the two bacteria and showed that the equilibrium bacterial count of S. aureus was the more sensitive parameter. The switch in growth rate happens at a pH of 6.5–7. Our studies are in line with the general hypothesis that keeping the skin pH within an acidic range is advantageous in terms of keeping the skin microbiome in balance and maintaining healthy skin.

Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device

2021 ◽  
Vol 70 (5) ◽  
Author(s):  
Adéla Diepoltová ◽  
Klára Konečná ◽  
Ondřej Janďourek ◽  
Petr Nachtigal
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Modification ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Type Species ◽  
Cultivation Conditions ◽  
Content Type ◽  
Link Type ◽  
The Impact

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections. Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results. Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied. Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis. Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain. Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).

Eikenella glucosivorans sp. nov., isolated from a human throat swab, and emendation of the genus Eikenella to include saccharolytic species

2021 ◽  
Vol 71 (9) ◽  
Author(s):  
Silvio Hering ◽  
Moritz K. Jansson ◽  
Michael E. J. Buhl
Keyword(s):  
Type Species ◽  
Throat Swab ◽  
Novel Species ◽  
Phylogenetic Analyses ◽  
Routine Care ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Bacterium Strain

A novel species within the genus Eikenella is described, based on the phenotypical, biochemical and genetic characterization of a strain of a facultatively anaerobic, Gram-negative rod-shaped bacterium. Strain S3360T was isolated from the throat swab of a patient sampled during routine care at a hospital. Phylogenetic analyses (full-length 16S rRNA gene and whole-genome sequences) placed the strain in the genus Eikenella , separate from all recognized species but with the closest relationship to Eikenella longinqua (NML 02-A-017T). Eikenella is one of the genera in the HACEK group known to be responsible for rare cases of endocarditis in humans. Until the recent descriptions of Eikenella exigua , Eikenella halliae and Eikenella longinqua , Eikenella corrodens had been the only validly published species in this genus since its description as Bacteroides corrodens in 1958. Unlike these species, strain S3360T is able to metabolize carbohydrates (glucose). The average nucleotide identities of strain S3360T with E. longinqua (NML 02-A-017T) and E. corrodens (NCTC 10596T), the type species of the genus, were 90.5 and 84.7 %, respectively, and the corresponding genome-to-genome distance values were 41.3 and 29.0 %, respectively. The DNA G+C content of strain S3360T was 58.4 mol%. Based on the phenotypical, biochemical and genetic findings, strain S3360T is considered to represent a novel species within the genus Eikenella , for which the name Eikenella glucosivorans sp. nov. is proposed. The type strain is S3360T (DSM 110714T=CCOS 1935T=CCUG 74293T). In addition, an emendation of the genus Eikenella is proposed to include species which are saccharolytic.

scholarly journals Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Kay Fountain ◽  
Tiffany Blackett ◽  
Helen Butler ◽  
Catherine Carchedi ◽  
Anna-Katarina Schilling ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
Small Ruminants ◽  
Sciurus Vulgaris ◽  
Red Squirrels ◽  
Content Type ◽  
Island Populations ◽  
Link Type ◽  
Bacterial Cell Membrane ◽  
Reservoir Hosts

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat (‘spillover’), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

scholarly journals Comparative genomics of Staphylococcus epidermidis from prosthetic-joint infections and nares highlights genetic traits associated with antimicrobial resistance, not virulence

2021 ◽  
Author(s):  
Emeli Månsson ◽  
Thor Bech Johannesen ◽  
Åsa Nilsdotter-Augustinsson ◽  
Bo Söderquist ◽  
Marc Stegger
Keyword(s):  
Population Structure ◽  
Antimicrobial Resistance ◽  
Staphylococcus Epidermidis ◽  
Type Species ◽  
Genetic Traits ◽  
Content Type ◽  
Link Type ◽  
Joint Infections ◽  
Prosthetic Joint ◽  
Prosthetic Joint Infections

There is increased awareness of the worldwide spread of specific epidemic multidrug-resistant (MDR) lineages of the human commensal Staphylococcus epidermidis . Here, using bioinformatic analyses accounting for population structure, we determined genomic traits (genes, SNPs and k-mers) that distinguish S. epidermidis causing prosthetic-joint infections (PJIs) from commensal isolates from nares, by analysing whole-genome sequencing data from S. epidermidis from PJIs prospectively collected over 10 years in Sweden, and contemporary S. epidermidis from the nares of patients scheduled for arthroplasty surgery. Previously suggested virulence determinants and the presence of genes and mutations linked to antimicrobial resistance (AMR) were also investigated. Publicly available S. epidermidis sequences were used for international extrapolation and validation of findings. Our data show that S. epidermidis causing PJIs differed from nasal isolates not by virulence but by traits associated with resistance to compounds used in prevention of PJIs: β-lactams, aminoglycosides and chlorhexidine. Almost a quarter of the PJI isolates did not belong to any of the previously described major nosocomial lineages, but the AMR-related traits were also over-represented in these isolates, as well as in international S. epidermidis isolates originating from PJIs. Genes previously associated with virulence in S. epidermidis were over-represented in individual lineages, but failed to reach statistical significance when adjusted for population structure. Our findings suggest that the current strategies for prevention of PJIs select for nosocomial MDR S. epidermidis lineages that have arisen from horizontal gene transfer of AMR-related traits into multiple genetic backgrounds.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Staphylococcus singaporensis sp. nov., a new member of the Staphylococcus aureus complex, isolated from human clinical specimens

2021 ◽  
Vol 71 (10) ◽  
Author(s):  
Ka Lip Chew ◽  
Sophie Octavia ◽  
Deborah Lai ◽  
Raymond T. P. Lin ◽  
Jeanette W. P. Teo
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
Clinical Laboratory ◽  
Core Gene ◽  
Separate Species ◽  
New Members ◽  
Content Type ◽  
Link Type ◽  
Flight Mass Spectrometry ◽  
Genotypic Analysis

Staphylococcus argenteus and Staphylococcus schweitzeri are the newest members of the Staphylococcus aureus complex. The number of clinical reports attributed to these new S. aureus complex members is limited. In a retrospective clinical laboratory study conducted over a 4-month period investigating the prevalence of S. argenteus and S. schweitzeri , a total of 43 isolates were selected. Phylogeny based on core-gene multilocus sequence typing (MLST) analysis confirmed that 37 were S. argenteus but a genetically distinct clade of six isolates was identified. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses further supported the classification of these six isolates as a separate species. When compared to S. aureus complex reference genomes, the ANI values were ≤94 % and the dDDH values were <53 %. Based on the seven-gene S. aureus MLST scheme, the six isolates belong to five novel allelic profiles (ST6105, ST6106, ST6107, ST6108 and ST109). Their clinical infection features were similar to S. aureus . Skin and soft tissue infections presented in four out of the six cases. Routine clinical diagnostic identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biochemical profiling does not differentiate these new members from the rest of the complex. Genotypic analysis suggests that the six isolates belong to a novel species, Staphylococcus singaporensis sp. nov. with isolate SS21T (=DSM 111408T=NCTC14419T) designated as the type strain.

Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015–2016

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Mark Philip Bugayong ◽  
Hidemasa Izumiya ◽  
Josie M. Bilar ◽  
Masatomo Morita ◽  
Eiji Arakawa ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Type Species ◽  
Variable Number Tandem Repeat ◽  
The Philippines ◽  
Large Chromosome ◽  
Content Type ◽  
Link Type ◽  
Mlva Type ◽  
El Tor

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

scholarly journals Mycobacterium celeriflavum sp. nov., a rapidly growing scotochromogenic bacterium isolated from clinical specimens

2015 ◽  
Vol 65 (Pt_2) ◽  
pp. 510-515 ◽  
Author(s):  
Abdolrazagh Hashemi Shahraki ◽  
Cengiz Çavuşoğlu ◽  
Emanuele Borroni ◽  
Parvin Heidarieh ◽  
Orhan Kaya Koksalan ◽  
...  
Keyword(s):  
Type Strain ◽  
Type Species ◽  
Sequence Similarity ◽  
Housekeeping Genes ◽  
Mycolic Acid ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Polyphasic Characterization

Six strains of a rapidly growing scotochromogenic mycobacterium were isolated from pulmonary specimens of independent patients. Biochemical and cultural tests were not suitable for their identification. The mycolic acid pattern analysed by HPLC was different from that of any other mycobacterium. Genotypic characterization, targeting seven housekeeping genes, revealed the presence of microheterogeneity in all of them. Different species were more closely related to the test strains in various regions: the type strain of Mycobacterium moriokaense showed 99.0 % 16S rRNA gene sequence similarity, and 91.5–96.5 % similarity for the remaining six regions. The whole genome sequences of the proposed type strain and that of M. moriokaense presented an average nucleotide identity (ANI) of 82.9 %. Phylogenetic analysis produced poorly robust trees in most genes with the exception of rpoB and sodA where Mycobacterium flavescens and Mycobacterium novocastrense were the closest species. This phylogenetic relatedness was confirmed by the tree inferred from five concatenated genes, which was very robust. The polyphasic characterization of the test strains, supported by the ANI value, demonstrates that they belong to a previously unreported species, for which the name Mycobacterium celeriflavum sp. nov. is proposed. The type strain is AFPC-000207T ( = DSM 46765T = JCM 18439T).

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