PBP4: A New Perspective on Staphylococcus aureus β-Lactam Resistance.

β-lactam antibiotics are excellent drugs for treatment of staphylococcal infections, due to their superior efficacy and safety compared to other drugs. Effectiveness of β-lactams is severely compromised due to resistance, which is widespread among clinical strains of Staphylococcus aureus. β-lactams inhibit bacterial cells by binding to penicillin binding proteins (PBPs), which perform the penultimate steps of bacterial cell wall synthesis. Among PBPs of S. aureus, PBP2a has received the most attention for the past several decades due to its preeminent role in conferring both high-level and broad-spectrum resistance to the entire class of β-lactam drugs. Studies on PBP2a have thus unraveled incredible details of its mechanism of action. We have recently identified that an uncanonical, low molecular weight PBP of S. aureus, PBP4, can also provide high-level and broad-spectrum resistance to the entire class of β-lactam drugs at a level similar to that of PBP2a. The role of PBP4 has typically been considered not so important for β-lactam resistance of S. aureus, and as a result its mode of action remains largely unknown. In this article, we review our current knowledge of PBP4 mediating β-lactam resistance in S. aureus

Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

<p>Abstract</p> <p>Background</p> <p>The opportunistic food-borne gram-positive pathogen <it>Listeria monocytogenes </it>can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σ^B, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σ^Bexpression during growth of <it>L. monocytogenes </it>EGD-e. Whole genome-based transcriptional profiling was used to identify σ^B-dependent genes at different growth phases.</p> <p>Results</p> <p>We detected 105 σ^B-positively regulated genes and 111 genes which appeared to be under negative control of σ^Band validated 36 σ^B-positively regulated genes <it>in vivo </it>using a reporter gene fusion system.</p> <p>Conclusion</p> <p>Genes comprising the σ^Bregulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σ^B-positively regulated genes of <it>L. monocytogenes </it>are absent in <it>L. innocua</it>. A total of 75 σ^B-positively regulated listerial genes had homologs in <it>B. subtilis</it>, but only 33 have been previously described as being σ^B-regulated in <it>B. subtilis </it>even though both species share a highly conserved σ^B-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.</p

Distribution and Regulation of the Mobile Genetic Element-Encoded Phenol-Soluble Modulin PSM-mec in Methicillin-Resistant Staphylococcus aureus

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus

Complete genome sequence of a Pantón-Valentine leukocidin-negative community-associated methicillin-resistant Staphylococcus aureus strain of sequence type 72 from Korea.

In the past decade, community-associated (CA-) infections with methicillin-resistant Staphylococcus aureus (MRSA) have emerged throughout the world. Different CA-MRSA strains dominate in different geographical locations. Many CA-MRSA lineages contain genes coding for the Pantón-Valentine leukocidin. However, the role of this leukotoxin in CA-MRSA pathogenesis is still controversial. The genome sequences of two key PVL-positive CA-MRSA strains (USA300, USA400) have been reported, but we lack information on the more recently found PVL-negative CA-MRSA strains. One such strain is the PVL-negative ST72, the main cause of CA-MRSA infections in Korea. Here, we report the entire genome sequence of CA-MRSA ST72 and analyze its gene content with a focus on virulence factors. Our results show that this strain does not have considerable differences in virulence factor content compared to other CA-MRSA strains (USA300, USA400), indicating that other toxins do not substitute for the lack of PVL in ST72. This finding is in accordance with the notion that differential expression of widespread virulence determinants, rather than the acquisition of additional virulence factors on mobile genetic elements, such as PVL, is responsible for the increased virulence of CA- compared to hospital-associated MRSA

Ceftobiprole- and Ceftaroline-Resistant Methicillin-Resistant Staphylococcus aureus

The role of mecA mutations in conferring resistance to ceftobiprole and ceftaroline, cephalosporins with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, was determined with MRSA strains COL and SF8300. The SF8300 ceftaroline-passaged mutant carried a single mecA mutation, E447K (E-to-K change at position 447), and expressed low-level resistance. This mutation in COL conferred high-level resistance to ceftobiprole but only low-level resistance to ceftaroline. The COL ceftaroline-passaged mutant, which expressed high-level resistance to ceftobiprole and ceftaroline, had mutations in pbp2, pbp4, and gdpP but not mecA

Ceftobiprole- and Ceftaroline-Resistant Methicillin-Resistant Staphylococcus aureus

The role of mecA mutations in conferring resistance to ceftobiprole and ceftaroline, cephalosporins with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, was determined with MRSA strains COL and SF8300. The SF8300 ceftaroline-passaged mutant carried a single mecA mutation, E447K (E-to-K change at position 447), and expressed low-level resistance. This mutation in COL conferred high-level resistance to ceftobiprole but only low-level resistance to ceftaroline. The COL ceftaroline-passaged mutant, which expressed high-level resistance to ceftobiprole and ceftaroline, had mutations in pbp2, pbp4, and gdpP but not mecA

Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Resistant to Fifth-Generation Cephalosporins Reveals Potential Non-mecA Mechanisms of Resistance.

Fifth-generation cephalosporins, ceftobiprole and ceftaroline, are promising drugs for treatment of bacterial infections from methicillin-resistant Staphylococcus aureus (MRSA). These antibiotics are able to bind native PBP2a, the penicillin-binding protein encoded by the mecA resistance determinant that mediates broad class resistance to nearly all other beta-lactam antibiotics, at clinically achievable concentrations. Mechanisms of resistance to ceftaroline based on mecA mutations have been previously described. Here we compare the genomes of 11 total parent-daughter strains of Staphylococcus aureus for which specific selection by serial passaging with ceftaroline or ceftobiprole was used to identify novel non-mecA mechanisms of resistance. All 5 ceftaroline-resistant strains, derived from 5 different parental strains, contained mutations directly upstream of the pbp4 gene (coding for the PBP4 protein), including four with the same thymidine insertion located 377 nucleotides upstream of the promoter site. In 4 of 5 independent ceftaroline-driven selections, we also isolated mutations to the same residue (Asn138) in PBP4. In addition, mutations in additional candidate genes such as ClpX endopeptidase, PP2C protein phosphatase and transcription terminator Rho, previously undescribed in the context of resistance to ceftaroline or ceftobiprole, were detected in multiple selections. These genomic findings suggest that non-mecA mechanisms, while yet to be encountered in the clinical setting, may also be important in mediating resistance to 5th-generation cephalosporins