Characterization of compensatory mutations associated with restoration of daptomycin-susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus and the role mprF mutations.

The objective of this study was to investigate the underlying mechanism explaining reversion of clinical DAP non-susceptible (NS) MRSA isolates to DAP-susceptible (S) by analysis of genomic and cell wall characteristics of clinical DAP-NS MRSA and DAP-S MRSA isolates as well as in vitro revertant DAP-S MRSA using whole genome sequencing (WGS) and analysis of biological properties. WGS of the 4 clinical DAP-NS MRSA revealed mprF mutations resulting in amino acid substitutions or deletion. These same amino acid substitutions and deletion were also observed in the 4 in vitro revertant DAP-S strains. While WGS identified the presence of the same mprF mutations in both the DAP-NS and in vitro DAP-S revertant strains, new mutations were also detected in other genes and intergenic regions of in vitro DAP-S revertant strains. Transmission electron microscopy to assess cell-wall (CW) thickness of 4 sets strains (pre- and post-DAP therapy isolates and in vitro DAP-S revertant) showed that 3 of the 4 isolates developed increased thickness of the CW after DAP therapy. After reversion to DAP susceptibility, CW thickness was decreased to the same level as DAP-S MRSA. Our results indicate that in vitro conversion of DAP-NS MRSA to DAP-S is independent of mprF gene mutations and may be partially explained by a change in CW thickness. However, as some strains showed no change in the CW, further studies are required to elucidate the different mechanisms of resistance to DAP, and factors for conversion of DAP-NS to DAP-S.

Complete genome sequencing of three human clinical isolates of Staphylococcus caprae reveals virulence factors similar to those of S. epidermidis and S. capitis.

BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus.

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the ß subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.

"Slow VISA," a novel phenotype of vancomycin resistance, found in vitro in heterogeneous vancomycin-intermediate Staphylococcus aureus strain Mu3.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1×10(-6)). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase ß-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

A Comprehensive Review on Phage Therapy and Phage-Based Drug Development.

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices.

Determination of the three-dimensional structure of bacteriophage Mu(-) tail fiber and its characterization.

Bacteriophage Mu is a temperate phage known to infect various species of Enterobacteria, playing a role in bacterial mutation induction and horizontal gene transfer. The phage possesses two types of tail fibers important for host recognition, which enable it to expand its range of hosts. The alternate tail fibers are formed through the action of genes 49-50 or 52-51, allowing the Mu phage to recognize different surfaces of host cells. In a previous study, we presented the X-ray crystal structure of the C-terminal lipopolysaccharide (LPS)-binding domain of gene product (gp) 49, one of the subunits comprising the Mu tail fiber. In this study, we have determined the structure of the alternative tail fiber subunit, gp52, and compared it with other tail fibers. The results revealed that Mu phage employs different structural motifs for two individual tail fibers for recognizing different hosts.

Efficient synthesis of CRISPR-Cas13a-antimicrobial capsids against MRSA facilitated by silent mutation incorporation.

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Phagemid-based capsid system for CRISPR-Cas13a antimicrobials targeting methicillin-resistant Staphylococcus aureus.

In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Mutation of RNA polymerase ß-subunit gene promotes heterogeneous-to-homogeneous conversion of ß-lactam resistance in methicillin-resistant Staphylococcus aureus.

Three types of phenotypic expression of ß-lactam resistance have been reported in methicillin-resistant Staphylococcus aureus (MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of ß-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) in addition to the expression of the mecA gene. Eagle-type resistance is a unique phenotype of chr* occurring in pre-MRSA strain N315 whose mecA gene expression is strongly repressed by an intact mecI gene. We here report that certain mutations of the rpoB gene, encoding the RNA polymerase ß subunit, belong to chr*. We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of the rpoB(N967I) mutation was confirmed by constructing a revertant H5 rpoB(I967N) strain as well as an N315-derived mutant, N315 rpoB(N967I). H5 rpoB(I967N) regained the hetero-resistance phenotype, and the N315 rpoB(N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation of rpoB(R644H). Introduction of the rpoB(N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider that rpoB mutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of ß-lactam resistance in MRSA strain N315 and its derived strains.

Sphingobacterium thermophilum sp. nov., of the phylum Bacteroidetes, isolated from compost.

A Gram-negative bacterium, designated CKTN2(T), was isolated from compost. Cells of strain CKTN2(T) were strictly aerobic rods. The isolate grew at 20-50 °C (optimum 40-45 °C), but not below 15 °C or above 52 °C, and at pH 5.9-8.8 (optimum pH 7.0), but not below pH 5.4 or above pH 9.3. The DNA G+C content was 40.3 mol%. The predominant menaquinone was MK-7. The major fatty acids were iso-C15 : 0 (45.2 %), iso-C17 : 0 3-OH (11.1 %) and C18 : 0 (14.5 %). Analysis of the 16S rRNA gene sequence of strain CKTN2(T) revealed that it is a member of the genus Sphingobacterium and is most closely related to Sphingobacterium alimentarium DSM 22362(T) (93.2 % 16S rRNA gene sequence similarity). Strain CKTN2(T) could be distinguished from its closest phylogenetic relatives by different phenotypic characteristics. According to the phenotypic and genotypic characteristics, strain CKTN2(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium thermophilum sp. nov. is proposed. The type strain is CKTN2(T) ( = JCM 17858(T)  = KCTC 23708(T)).

CID12261165, a flavonoid compound as antibacterial agents against quinolone-resistant Staphylococcus aureus.

Flavonoids are plant-produced secondary metabolites that are found ubiquitously. We have previously reported that apigenin, a class of flavonoid, has unique antimicrobial activity against Staphylococcus aureus (S. aureus), one of the major human pathogens. Apigenin inhibited fluoroquinolone-resistant S. aureus with DNA gyrase harboring the quinolone-resistant S84L mutation but did not inhibit wild-type DNA gyrase. In this study, we describe five flavonoids, quercetin, luteolin, kaempferol, baicalein, and commercially available CID12261165, that show similar antimicrobial activity against fluoroquinolone-resistant S. aureus. Among them, CID12261165 was the most effective with MIC values of ≤ 4 mg/L against quinolone-resistant S. aureus strains. In vitro DNA cleavage and supercoiling assays demonstrated inhibitory activity of CID12261165 against mutated DNA gyrase, whereas activity against wild-type DNA gyrase was not observed. CID12261165 also inhibited quinolone-resistant Enterococci with an MIC value of 8 mg/L. While fluoroquinolone-resistant amino acid replacements can improve the fitness of bacterial cells, it is unknown why quinolone-susceptible S. aureus strains were predominant before the introduction of fluoroquinolone. The present study discusses the current discrepancies in the interpretation of antimicrobial activities of flavonoids, as well as the possible reasons for the preservation of wild-type DNA gyrase wherein the environmental flavonoids cannot be ignored.

Thermovum composti gen. nov., sp. nov., an alphaproteobacterium from compost.

A Gram-stain-positive thermophilic bacterium, designated strain Nis3(T), was isolated from compost. The strain grew at 23-57 °C (optimum, 50 °C); no growth was observed below 15 or above 60 °C. The pH range for growth was 5.9-8.8 (optimum, 7.0); no growth was observed below pH 5.4 or above pH 9.3. The DNA G+C content of strain Nis3(T) was 63.4 mol%. The dominant quinone type was ubiquinone Q-10. The major fatty acids were C(18:1)ω7c, C(19:0)ω8c cyclo and C(18:0). The polar lipids comprised phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylmonomethylethanolamine, an unknown glycolipid and a ninhydrin-positive phospholipid. 16S rRNA gene sequence analysis assigned this bacterium to the family Phyllobacteriaceae in the Alphaproteobacteria but it shared less than 95.2% sequence similarity with other members of the family. The chemotaxonomic and phenotypic characteristics of strain Nis3(T) differed in some respects from those of members of the family Phyllobacteriaceae. Therefore, strain Nis3(T) is considered to represent a novel species of a new genus in the family Phyllobacteriaceae, for which the name Thermovum composti gen. nov., sp. nov. is proposed. The type strain is Nis3(T) ( = JCM 17863(T) = KCTC 23707(T)).

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

Thermasporomyces composti gen. nov., sp. nov., a thermophilic actinomycete isolated from compost.

A thermophilic, Gram-positive bacterium that formed a branched vegetative mycelium was isolated from compost. The strain, designated I3(T), grew at temperatures between 35 and 62 °C, with optimum growth at 50-55 °C. No growth was observed below 29 °C or above 65 °C. The pH range for growth was 5.7-10.0, the pH for optimum growth was 7.0 and no growth was observed below pH 5.6 or above pH 10.8. The DNA G+C content of strain I3(T) was 69.2 mol%. The major fatty acids found were C(15 : 0) iso (14.2 %), C(15 : 0) anteiso (12.1 %), C(17 : 0) iso (16.3 %) and C(17 : 0) anteiso (21.7 %). The major menaquinones were MK-9(H(4)), MK-10(H(4)) and MK-11(H(4)). The cell wall contained glutamic acid, glycine, alanine and ll-diaminopimelic acid in a molar ratio of 1.0 : 3.9 : 0.6 : 0.5. The polar lipids consisted of ninhydrin-positive phosphoglycolipids, phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid. The cell-wall sugars were rhamnose and arabinose. 16S rRNA gene sequence analysis assigned this actinomycete to the family Nocardioidaceae, but its 16S rRNA gene sequence shared no more than 95.5 % similarity with those of other members of the family. The chemotaxonomic and phenotypic characteristics of strain I3(T) differed in some respects from those of members of the genus Actinopolymorpha, the most closely related genus. Therefore, strain I3(T) represents a novel species in a new genus of the family Nocardioidaceae, for which the name Thermasporomyces composti gen. nov., sp. nov. is proposed. The type strain of the type species is I3(T) (=JCM 16421(T)=DSM 22891(T)).

Thermogemmatispora onikobensis gen. nov., sp. nov. and Thermogemmatispora foliorum sp. nov., isolated from fallen leaves on geothermal soils, and description of Thermogemmatisporaceae fam. nov. and Thermogemmatisporales ord. nov. within the class Ktedonobacteria.

Two thermophilic, Gram-stain-positive, sporulating bacterial strains, which formed branched vegetative and aerial mycelia, were isolated from fallen leaves sampled from geothermal soils and designated ONI-1(T) and ONI-5(T). Strain ONI-1(T) grew at 50-74 °C, with optimum growth at 60-65 °C, and strain ONI-5(T) grew at 45-74 °C, with optimum growth at 60-65 °C. The pH range for growth of the strains was pH 4.6-8.0, with optimum growth at pH 7.0. The DNA G+C contents of strains ONI-1(T) and ONI-5(T) were 60.2 and 58.1 mol%, respectively. The major fatty acid was iso-C(17 : 0) and the major menaquinone was MK-9(H(2)). The cell walls of the strains contained glutamic acid, serine, glycine, histidine, alanine and ornithine. The polar lipids consisted of phosphatidylinositol, phosphatidylglycerol and a glycolipid. The cell-wall sugar was rhamnose. Detailed phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains belong to the class Ktedonobacteria and that strains ONI-1(T) and ONI-5(T) are most closely related to Thermosporothrix hazakensis SK20-1(T) (85.3 and 84.5 % sequence similarity, respectively). 16S rRNA gene sequence similarity between the two strains was 96.6 %. Based on the phenotypic features and phylogenetic position, we propose that strains ONI-1(T) and ONI-5(T) constitute a novel genus containing two novel species, for which we propose the names Thermogemmatispora onikobensis gen. nov., sp. nov. (the type species; type strain ONI-1(T)  = JCM 16817(T)  = KCTC 19768(T)) and Thermogemmatispora foliorum sp. nov. (type strain ONI-5(T)  = JCM 16818(T)  = KCTC 19767(T)), within the new family Thermogemmatisporaceae fam. nov. and order Thermogemmatisporales ord. nov.

A life cycle of branched aerial mycelium- and multiple budding spore-forming bacterium Thermosporothrix hazakensis belonging to the phylum Chloroflexi.

As far as known, sporulation modes in prokaryotes include formation of endospores exemplified by Firmicutes bacteria, myxospores by myxobacteria and arthrospores by actinomycetes. Here we describe Thermosporothrix hazakensis strain SK20-1(T) belonging to the phylum Chloroflexi with a life cycle including a novel prokaryotic sporulation mode. Microscopic observations showed that strain SK20-1(T) formed multiple exospores per mother cell by budding in branched aerial mycelia. Although branched aerial mycelia are characteristic of actinomycetes, multiple budding sporulation has not been previously described in prokaryotes. The strain SK20-1(T) could be a model microbe for cellular differentiation with multiple budding spore formation.

Association of mprF mutations with cross-resistance to daptomycin and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA).

We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.

Corrigendum: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

[This corrects the article DOI: 10.3389/fmicb.2019.02838.].

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.