SCCmec transformation requires living donor cells in mixed biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that has emerged through the horizontal acquisition of the staphylococcal cassette chromosome mec (SCCmec). Previously, we showed that SCCmec from heat-killed donors can be transferred via natural transformation in biofilms at frequencies of 10-8-10-7. Here, we show an improved transformation assay of SCCmec with frequencies up to 10-2 using co-cultured biofilms with living donor cells. The Ccr-attB system played an important role in SCCmec transfer, and the deletion of ccrAB recombinase genes reduced the frequency ∼30-fold. SCCmec could be transferred from either MRSA or methicillin-resistant coagulase-negative staphylococci to some methicillin-sensitive S. aureus recipients. In addition, the transformation of other plasmid or chromosomal genes is enhanced by using living donor cells. This study emphasizes the role of natural transformation as an evolutionary ability of S. aureus and in MRSA emergence.

Novel multicellular prokaryote discovered next to an underground stream.

A diversity of prokaryotes currently exhibit multicellularity with different generation mechanisms in a variety of contexts of ecology on Earth. In the present study, we report a new type of multicellular bacterium, HS-3, isolated from an underground stream. HS-3 self-organizes its filamentous cells into a layer-structured colony with the properties of a nematic liquid crystal. After maturation, the colony starts to form a semi-closed sphere accommodating clusters of coccobacillus daughter cells and selectively releases them upon contact with water. This is the first report that shows that a liquid-crystal status of cells can support the prokaryotic multicellular behavior. Importantly, the observed behavior of HS-3 suggests that the recurrent intermittent exposure of colonies to water flow in the cave might have been the ecological context that cultivated the evolutionary transition from unicellular to multicellular life. This is the new extant model that underpins theories regarding a role of ecological context in the emergence of multicellularity.

Trend in healthcare-associated infections due to vancomycin-resistant Enterococcus at a hospital in the era of COVID-19: More than hand hygiene is needed.

BACKGROUND: Variable control measures for vancomycin-resistant Enterococcus (VRE) infections were adopted among different hospitals and areas. We investigated the burden and patient characteristics of healthcare-associated VRE infections in 2018-2019 and 2020, when multiple preventive measures for COVID-19 were taken. METHODS: During the COVID-19 pandemic, mask waring and hand hygiene were enforced in the study hospital. The incidence densities of healthcare-associated infections (HAIs), including overall HAIs, methicillin-resistant Staphylococcus aureus (MRSA) HAIs, VRE HAIs, and VRE healthcare-associated bloodstream infections (HABSIs), consumption of broad-spectrum antibiotics and hygiene products, demographic characteristics and medical conditions of affected patients, were compared before and after the pandemic. RESULTS: The incidence density of both VRE HAIs and VRE HABSIs did not change statistically significantly, however, the highest in 2020 than that in 2018 and 2019. This was in spite of universal mask waring and increased consumption of 75% alcohol in 2020 and consistent implementation of an antibiotic stewardship program in three observed years. The increased prescriptions of broad-spectrum cephalosporins might partially explain the increase of VRE infection. CONCLUSION: Increased mask wearing and hand hygiene may not result in the decline in the development of VRE HAIs in the hospital during the COVID-19 pandemic, and continued monitoring of the dynamics of HAIs remains indispensable.

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms.

SCCmec is a large mobile genetic element that includes the mecA gene and confers resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA). There is evidence that SCCmec disseminates among staphylococci, but the transfer mechanisms are unclear. Here, we show that two-component systems mediate the upregulation of natural competence genes in S. aureus under biofilm growth conditions, and this enhances the efficiency of natural transformation. We observe SCCmec transfer via natural transformation from MRSA, and from methicillin-resistant coagulase-negative staphylococci, to methicillin-sensitive S. aureus. The process requires the SCCmec recombinase genes ccrAB, and the stability of the transferred SCCmec varies depending on SCCmec types and recipients. Our results suggest that natural transformation plays a role in the transfer of SCCmec and possibly other mobile genetic elements in S. aureus biofilms.

No Change, No Life? What We Know about Phase Variation in Staphylococcus aureus.

Phase variation (PV) is a well-known phenomenon of high-frequency reversible gene-expression switching. PV arises from genetic and epigenetic mechanisms and confers a range of benefits to bacteria, constituting both an innate immune strategy to infection from bacteriophages as well as an adaptation strategy within an infected host. PV has been well-characterized in numerous bacterial species; however, there is limited direct evidence of PV in the human opportunistic pathogen Staphylococcus aureus. This review provides an overview of the mechanisms that generate PV and focuses on earlier and recent findings of PV in S. aureus, with a brief look at the future of the field.

The emergence of plasmid-borne cfr-mediated linezolid resistant-staphylococci in Vietnam.

OBJECTIVES: Linezolid is one of the last resort antibiotics effectively used in the treatment of infections caused by multidrug-resistant Gram-positive bacteria. Recent outbreaks of Linezolid resistance have been the great concern worldwide, while many countries have not experienced it. In this work, we aimed to evaluate the existence of linezolid resistance and further clarify potential resistance mechanism(s) in staphylococcal isolates obtained from the hospital in Vietnam, a country in which linezolid resistance had not been previously detected. METHODS: Seventy staphylococcal clinical isolates including MRSA (n=63) and methicillin-resistant coagulase-negative staphylococci (MRCNS, n=7) were collected and analyzed for linezolid resistance. Linezolid-resistant isolates were submitted for whole genome sequencing to search for the resistance determinants. RESULTS: We identified two coagulase-negative staphylococcal isolates that were resistant to linezolid. Whole genome sequencing revealed several alterations in the 23S rRNA and L3, L17, L22, L24, L30 ribosomal proteins. Importantly, both isolates harbour the chloramphenicol/florfenicol resistance (cfr) gene on a plasmid. The plasmid was closely identical to the pLRSA417 plasmid that was originally reported in China. CONCLUSIONS: To the best of our knowledge, this is the first report of cfr-mediated linezolid resistance in clinically isolated staphylococci in Vietnam. We suggest that adequate surveillance is necessary to monitor the dissemination of linezolid resistance among staphylococcal species and other important pathogens.

What Happens in the Staphylococcal Nucleoid under Oxidative Stress?

The evolutionary success of Staphylococcus aureus as an opportunistic human pathogen is largely attributed to its prominent abilities to cope with a variety of stresses and host bactericidal factors. Reactive oxygen species are important weapons in the host arsenal that inactivate phagocytosed pathogens, but S. aureus can survive in phagosomes and escape from phagocytic cells to establish infections. Molecular genetic analyses combined with atomic force microscopy have revealed that the MrgA protein (part of the Dps family of proteins) is induced specifically in response to oxidative stress and converts the nucleoid from the fibrous to the clogged state. This review collates a series of evidences on the staphylococcal nucleoid dynamics under oxidative stress, which is functionally and physically distinct from compacted Escherichia coli nucleoid under stationary phase. In addition, potential new roles of nucleoid clogging in the staphylococcal life cycle will be proposed.

Finding of Agr Phase Variants in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.IMPORTANCEStaphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment.

Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings.

Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization.

This protocol describes the application of atomic force microscopy for structural analysis of the prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables step-wise dissection of the nucleoid. The procedure includes (1) on-substrate-lysis of cells, and (2) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural re-organization or building up of basic structures, as seen for Dps in Escherichia coli, and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.

Probiotics into outer space: feasibility assessments of encapsulated freeze-dried probiotics during 1 month's storage on the International Space Station.

Suppression of immune function during long spaceflights is an issue that needs to be overcome. The well-established probiotic Lactobacillus casei strain Shirota (LcS) could be a promising countermeasure, and we have launched a project to investigate the efficacy of its use on the International Space Station (ISS). As a first step, we developed a specialist probiotic product for space experiments, containing freeze-dried LcS in capsule form (Probiotics Package), and tested its stability through 1 month of storage on the ISS. The temperature inside the ISS ranged from 20.0 to 24.5 °C. The absorbed dose rate of the flight sample was 0.26 mGy/day and the dose equivalent rate was 0.52 mSv/day. The number of live LcS was 1.05 × 1011 colony-forming units/g powder (49.5% of the initial value) 6 months after the start of the study; this value was comparable to those in the two ground controls. Profiles of randomly amplified polymorphic DNA, sequence variant frequency, carbohydrate fermentation, reactivity to LcS-specific antibody, and the cytokine-inducing ability of LcS in the flight sample did not differ from those of the ground controls. We can therefore maintain the viability and basic probiotic properties of LcS stored as a Probiotics Package on the ISS.

Sodium Polyanethol Sulfonate Modulates Natural Transformation of SigH-Expressing Staphylococcus aureus.

Expression of genes required for natural genetic competence in Staphylococcus aureus is controlled by an alternative transcription sigma factor, SigH. However, even in the SigH-expressing cells, the DNA transformation efficiency varies depending on culture conditions. We report here that cells grown in the competence-inducing medium (CS2 medium) exhibit enlarged morphology with disintegrated cell walls. Notably, an autolysis inhibitor, Sodium Polyanethol Sulfonate (SPS), facilitated transformation in CS2 medium in a dose-dependent manner, suggesting the involvement of the cell wall metabolism in transformation. However, the transformation efficiency of cells grown in TSB was not improved by physical or enzymatic damage on the cell walls.

Cellular splicing factor UAP56 stimulates trimeric NP formation for assembly of functional influenza viral ribonucleoprotein complexes.

The influenza virus RNA genome exists as a ribonucleoprotein (RNP) complex by interacting with NP, one of virus-encoded RNA binding proteins. It is proposed that trimeric NP is a functional form, but it is not clear how trimeric NP is formed and transferred to RNA. UAP56, a cellular splicing factor, functions as a molecular chaperone for NP and is required for the replication-coupled RNP formation of newly synthesized viral genome, but the details of NP transfer to viral RNA by UAP56 is unclear. Here we found that UAP56 is complexed with trimeric NP, but not monomeric NP. Gel filtration analysis and atomic force microscopy analysis indicated that the complex consists of two trimeric NP connected by UAP56. We also found that UAP56 stimulates trimeric NP formation from monomeric NP even at physiological salt concentrations. Thus, UAP56 facilitates the transfer of NP to viral RNA since trimeric NP has higher RNA binding activity than monomeric NP. Further, UAP56 represses the binding of excess amount of NP to RNA possibly by transferring trimeric NP. Collectively, we propose that UAP56 stimulates viral RNP formation through promotion of the assembly of trimeric NP and is important for the structural integrity of NP-RNA complex.

Identification of nucleoid associated proteins (NAPs) under oxidative stress in Staphylococcus aureus.

BACKGROUND: Bacterial nucleoid consists of genome DNA, RNA, and hundreds of nucleoid-associated proteins (NAPs). Escherichia coli nucleoid is compacted towards the stationary phase, replacing most log-phase NAPs with the major stationary-phase nucleoid protein, Dps. In contrast, Staphylococcus aureus nucleoid sustains the fiber structures throughout the growth. Instead, the Dps homologue, MrgA, expresses under oxidative stress conditions to clump the nucleoid, but the composition of the clumped nucleoid was elusive. RESULTS: The staphylococcal nucleoid under oxidative stress was isolated by sucrose gradient centrifugation, and the proteins were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 299 proteins in the nucleoid under oxidative stress, including 113 csNAPs (contaminant-subtracted NAPs). Comparison with the previously identified csNAPs in log- and stationary phase indicated that one fifth of the csNAPs under oxidative stress were the constitutive nucleoid components; importantly, several factors including HU, SarA, FabZ, and ribosomes were sustained under oxidative stress. Some factors (e.g. SA1663 and SA0092/SA0093) with unknown functions were included in the csNAPs list specifically under oxidative stress condition. CONCLUSION: Nucleoid constitutively holds Hu, SarA, FabG, and ribosomal proteins even under the oxidative stress, reflecting the active functions of the clumped nucleoid, unlikely to the dormant E. coli nucleoid compacted in the stationary phase or starvation.

Adjacent-possible ecological niche: growth of Lactobacillus species co-cultured with Escherichia coli in a synthetic minimal medium.

In certain conditions, members of the Lactobacillus genus are auxotrophs that have fastidious requirements for growth. Notably, Lactobacillus cannot grow in M9 medium, a minimal synthetic medium used for Escherichia coli. However, we found that some Lactobacillus strains can be grown in M9 when co-cultured with E. coli K-12. In the co-culture, L. casei proliferates exponentially, reaching cell densities of 108 CFU (colony-forming unit) ml-1 in 6 h and dominating E. coli in the late growth phase. Spent medium from E. coli grown overnight lacked this growth-promoting effect on L. casei. Similarly, the effect was not observed when the species were separated by a 0.4-µm membrane. Microscopic observations showed that L. casei are embedded in the micro-scale clusters of E. coli in the early growth phase. This study describes for the first time the ability of a Lactobacillus species to grow in minimal medium when in close proximity with co-cultured bacteria.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Nucleoid clumping is dispensable for the Dps-dependent hydrogen peroxide resistance in Staphylococcus aureus.

Dps family proteins have the ferroxidase activity that contributes to oxidative stress resistance. In addition, a part of Dps family proteins including Escherichia coli Dps and Staphylococcus aureus MrgA (metallo regulon gene A) bind DNA and induce the structural change of the nucleoid. We previously showed that a mutated MrgA with reduced ferroxidase activity was unable to contribute to the hydrogen peroxide (H2O2) and UV resistance in S. aureus, suggesting that the nucleoid clumping by MrgA is not sufficient for the resistance. However, it remained elusive whether the nucleoid clumping is dispensable for the resistance. Here, we aimed to clarify this question by employing the E. coli Dps lacking DNA-binding activity, DpsΔ18. Staphylococcal nucleoid was clumped by E. coli Dps, but not by DpsΔ18. H2O2 stress assay indicated that Dps and DpsΔ18 restored the reduced susceptibility of S. aureus ΔmrgA. Thus, we concluded that the staphylococcal nucleoid clumping is dispensable for the Dps-mediated H2O2 resistance. In contrast, Dps was unable to complement S. aureus ΔmrgA in the UV resistance, suggesting the MrgA function that cannot be compensated for by E. coli Dps.

Listeria monocytogenes σH Contributes to Expression of Competence Genes and Intracellular Growth.

UNLABELLED: The alternative sigma factor σ(H)has two functions in Gram-positive bacteria: it regulates sporulation and the development of genetic competence. Listeria monocytogenes is a nonsporulating species in which competence has not yet been detected. Nevertheless, the main competence regulators and a series of orthologous genes that form the competence machinery are present in its genome; some of the competence genes play a role in optimal phagosomal escape. In this study, strains overexpressing σ(H) and strains with a σ(H) deletion were used to elucidate the contribution of σ(H) to the expression of the competence machinery genes inL. monocytogenes Gene expression analysis showed that σ(H) is, indeed, involved in comG and come regulation. Unexpectedly, we observed a unique regulation scheme in which σ(H) and the transcription factor ComK were involved. Population-level analysis showed that even with the overexpression of both factors, only a fraction of the cells expressed the competence machinery genes. Although we could not detect competence, σ(H) was crucial for phagosomal escape, which implies that this alternative sigma factor has specifically evolved to regulate the L. monocytogenes intracellular life cycle. IMPORTANCE: Listeria monocytogenes can be an intracellular pathogen capable of causing serious infections in humans and animal species. Recently, the competence machinery genes were described as being necessary for optimal phagosomal escape, in which the transcription factor ComK plays an important role. On the other hand, our previous phylogenetic analysis suggested that the alternative sigma factor σ(H) might play a role in the regulation of competence genes. The present study shows that some of the competence genes belong to the σ(H) regulon and, importantly, that σ(H) is essential for intracellular growth, implying a unique physiological role of σ(H) among Firmicutes.

Horizontal gene transmission of the cfr gene to MRSA and Enterococcus: role of Staphylococcus epidermidis as a reservoir and alternative pathway for the spread of linezolid resistance.

OBJECTIVES: Linezolid resistance mediated by the cfr gene represents a global concern due to its dissemination among multiresistant nosocomial pathogens such as MRSA and Enterococcus. In the present work, we have evaluated the in vitro transmission of cfr pSCFS7-like plasmids from two Staphylococcus epidermidis ST2 strains (SE45 and SE50) isolated in Spanish hospitals, to clinical MRSA and Enterococcus spp. isolates obtained in Japan, a country in which cfr has not been detected yet. We have also investigated alternative mechanisms of horizontal gene transfer involved in the spread of the cfr gene. METHODS: MRSA (n = 16) and Enterococcus spp. (n = 8) clinical isolates were used as recipients in conjugative experiments. Bacteriophage-mediated transmission was tested using MR83a phage and N315, COL and Mu50 strains. A transformation assay was carried out using a natural competent strain derived from N315. RESULTS: The SE45 strain was able to transfer the cfr gene to all strains tested, while transmission from SE50 was observed only to a few strains and with less efficiency. No transmission was observed to Enterococcus spp. isolates. Even though conjugation is thought to be the main mechanism of cfr dissemination, we have demonstrated that transduction can be considered an alternative pathway for transmission of the cfr gene between MRSA strains. However, the results suggest an absence of transmission by natural transformation. CONCLUSIONS: Linezolid resistance mediated by cfr vectors, such as pSCFS7-like plasmids, can be efficiently transferred to clinical MRSA in Japanese isolates. After reaching the staphylococcal pool, the cfr gene could be spread among MRSA strains by either conjugation or transduction.