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.

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.

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses.

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

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.

Identification and characterization of mutations responsible for the ß-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus.

Staphylococcus aureus strains that are susceptible to the ß-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire ß-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0-256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of ß-lactam resistance in OS-MRSA.