scholarly journals From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus

2021 ◽  
Author(s):  
Ian R. Monk ◽  
Timothy P. Stinear
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
Point Mutations ◽  
Site Specific ◽  
Content Type ◽  
Allelic Exchange ◽  
Link Type ◽  
Foreign Dna ◽  
Time Required ◽  
A Site

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.

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.

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.

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.

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.

scholarly journals Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms

Microbiology ◽  
2020 ◽  
Vol 166 (8) ◽  
pp. 695-706 ◽  
Author(s):  
Kevin H. Martin ◽  
Grace I. Borlee ◽  
William H. Wheat ◽  
Mary Jackson ◽  
Bradley R. Borlee
Keyword(s):  
Staphylococcus Aureus ◽  
Mycobacterium Bovis ◽  
Type Species ◽  
Acanthamoeba Castellanii ◽  
Methicillin Resistant ◽  
Content Type ◽  
Host Immune Responses ◽  
Link Type ◽  
Abiotic Surfaces ◽  
Low Concentrations

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae – Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum – and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis . The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

scholarly journals The general stress response of Staphylococcus aureus promotes tolerance of antibiotics and survival in whole human blood

Microbiology ◽  
2020 ◽  
Vol 166 (11) ◽  
pp. 1088-1094 ◽  
Author(s):  
Nisha Ranganathan ◽  
Rebecca Johnson ◽  
Andrew M. Edwards
Keyword(s):  
Staphylococcus Aureus ◽  
Stress Response ◽  
Human Blood ◽  
Chronic Infection ◽  
Type Species ◽  
Content Type ◽  
General Stress Response ◽  
Link Type ◽  
General Stress ◽  
Immune Defences

Staphylococcus aureus is a frequent cause of invasive human infections such as bacteraemia and infective endocarditis. These infections frequently relapse or become chronic, suggesting that the pathogen has mechanisms to tolerate the twin threats of therapeutic antibiotics and host immunity. The general stress response of S. aureus is regulated by the alternative sigma factor B (σB) and provides protection from multiple stresses including oxidative, acidic and heat. σB also contributes to virulence, intracellular persistence and chronic infection. However, the protective effect of σB on bacterial survival during exposure to antibiotics or host immune defences is poorly characterized. We found that σB promotes the survival of S. aureus exposed to the antibiotics gentamicin, ciprofloxacin, vancomycin and daptomycin, but not oxacillin or clindamycin. We also found that σB promoted staphylococcal survival in whole human blood, most likely via its contribution to oxidative stress resistance. Therefore, we conclude that the general stress response of S. aureus may contribute to the development of chronic infection by conferring tolerance to both antibiotics and host immune defences.

scholarly journals Molecular characterization of methicillin-resistant and -susceptible Staphylococcus aureus recovered from hospital personnel

2020 ◽  
Vol 69 (12) ◽  
pp. 1332-1338
Author(s):  
Zhen Xu ◽  
Xiaodong Li ◽  
Dan Tian ◽  
Zhuoyu Sun ◽  
Liqiong Guo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
North China ◽  
Molecular Characteristics ◽  
Hospital Personnel ◽  
Methicillin Resistant ◽  
Content Type ◽  
Link Type ◽  
Tertiary Hospitals ◽  
Genomic Studies

Introduction. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired infections. Over the past two decades MRSA has become ‘epidemic’ in many hospitals worldwide. However, little is known about the genetic background of S. aureus recovered from hospital personnel in China. Hypothesis/Gap Statement. The diversity of S. aureus genotypes warrants further surveillance and genomic studies to better understand the relatedness of these bacteria to those recovered from patients and the community. Aim. The aim of this study was to determine the genetic diversity of MRSA and methicillin-susceptible S. aureus (MSSA) recovered from hospital personnel in Tianjin, North China. Methodology. Three hundred and sixty-eight hand or nasal swabs were collected from 276 hospital personnel in 4 tertiary hospitals in Tianjin, North China between November 2017 and March 2019. In total, 535 Gram-positive bacteria were isolated, of which 59 were identified as S. aureus . Staphylococcal cassette chromosome mec (SCCmec) typing, multi-locus sequence typing (MLST) and spa typing were performed to determine the molecular characteristics of S. aureus . Results. Thirty-one out of 276 (11 %) hospital personnel were S. aureus carriers, whereas 11/276 (4 %) carried MRSA. Fifty out of 59 (85 %) S. aureus isolates were resistant or intermediately resistant to erythromycin. The dominant genotypes of MRSA recovered from hospital personnel were ST398-t034-SCCmecIV/V and ST630-t084/t2196, whereas the major genotypes of MSSA included ST15-t078/t084/t346/t796/t8862/t8945/t11653 and ST398-t189/t034/t078/t084/t14014. Conclusion. Although the predominant genotypes of MRSA recovered from hospital personnel in this study were different from the main genotypes that have previously been reported to cause infections in Tianjin and in other geographical areas of China, the MRSA ST398-t034 genotype has previously been reported to be associated with livestock globally. The dominant MSSA genotypes recovered from hospital personnel were consistent with the those previously reported to have been recovered from the clinic.

scholarly journals Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 837-848
Author(s):  
Yingyu Liu ◽  
Melanie J. Filiatrault
Keyword(s):  
Type Species ◽  
Plant Pathogens ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Soft Rot ◽  
Alternative Methods ◽  
Management Options ◽  
Content Type ◽  
Link Type ◽  
Resistant Mutants

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum -inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

scholarly journals Co-infection in critically ill patients with COVID-19: an observational cohort study from England

2021 ◽  
Vol 70 (4) ◽  
Author(s):  
Vadsala Baskaran ◽  
Hannah Lawrence ◽  
Louise E. Lansbury ◽  
Karmel Webb ◽  
Shahideh Safavi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cohort Study ◽  
Streptococcus Pneumoniae ◽  
Critically Ill ◽  
Type Species ◽  
Severe Illness ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Icu Stay

Introduction. During previous viral pandemics, reported co-infection rates and implicated pathogens have varied. In the 1918 influenza pandemic, a large proportion of severe illness and death was complicated by bacterial co-infection, predominantly Streptococcus pneumoniae and Staphylococcus aureus . Gap statement. A better understanding of the incidence of co-infection in patients with COVID-19 infection and the pathogens involved is necessary for effective antimicrobial stewardship. Aim. To describe the incidence and nature of co-infection in critically ill adults with COVID-19 infection in England. Methodology. A retrospective cohort study of adults with COVID-19 admitted to seven intensive care units (ICUs) in England up to 18 May 2020, was performed. Patients with completed ICU stays were included. The proportion and type of organisms were determined at <48 and >48 h following hospital admission, corresponding to community and hospital-acquired co-infections. Results. Of 254 patients studied (median age 59 years (IQR 49–69); 64.6 % male), 139 clinically significant organisms were identified from 83 (32.7 %) patients. Bacterial co-infections/ co-colonisation were identified within 48 h of admission in 14 (5.5 %) patients; the commonest pathogens were Staphylococcus aureus (four patients) and Streptococcus pneumoniae (two patients). The proportion of pathogens detected increased with duration of ICU stay, consisting largely of Gram-negative bacteria, particularly Klebsiella pneumoniae and Escherichia coli . The co-infection/ co-colonisation rate >48 h after admission was 27/1000 person-days (95 % CI 21.3–34.1). Patients with co-infections/ co-colonisation were more likely to die in ICU (crude OR 1.78,95 % CI 1.03–3.08, P=0.04) compared to those without co-infections/ co-colonisation. Conclusion. We found limited evidence for community-acquired bacterial co-infection in hospitalised adults with COVID-19, but a high rate of Gram-negative infection acquired during ICU stay.

Phenotypic and genetic characteristics of Streptococcus mutans isolates from site-specific dental plaque in China

2021 ◽  
Author(s):  
Shanshan Liu ◽  
Huihui Li ◽  
Kai Zhang ◽  
Zhenfei Guo ◽  
Qingwei Zheng ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Dental Plaque ◽  
Type Species ◽  
Single Subject ◽  
Genetic Characteristics ◽  
Site Specific ◽  
Content Type ◽  
Expression Levels ◽  
Link Type ◽  
Sequence Types

Introduction. Streptococcus mutans is an important cariogenic microbe. Hypothesis/Gap Statement. The potential characteristics of S. mutans isolates from site-specific dental plaque are still not clear. Aim. This study aimed to investigate the phenotypic and genetic characteristics of S. mutans isolates from site-specific dental plaque in China. Methodology. We used S. mutans isolated from children with early-childhood caries (ECC) and caries-free children to compare the phenotypic and genetic characteristics of S. mutans from site-specific dental plaque samples. The ECC subjects presented two sites: a cavitated lesion and a sound surface. The caries-free subjects presented one sound surface. Growth pattern, biofilm, decrease in pH, extracellular polysaccharide, expression levels of virulence-related genes, multilocus sequence typing (MLST) and phylogenetic trees were evaluated among these three sites. Results. The phenotypes detected between the cavitated and sound surfaces of ECC children were similar. However, the capacity for biofilm formation, pH drop and expression levels of genes (gtfB and spaP) of S. mutans in the caries-free group were lower compared with those of the ECC group. We identified 44 new alleles and 77 new sequence types. More than 90 % of the children with ECC shared an identical sequence type. The distribution of sequence types among different subjects showed diversity, and child-to-child transmission was detected. Conclusions. This is the first report of MLST on site-specific dental plaques in a single subject, and indicates that S. mutans isolated from site-specific dental plaque of a single subject showed similar phenotypes as a result of the isolates were closely related.

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