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

Bicarbonate Resensitization of Methicillin-Resistant Staphylococcus aureus to β-Lactam Antibiotics.

Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major health care concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as "resistant" to β-lactams, often leading to the use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied. We identified two distinct MRSA phenotypes upon AST using standard media, with or without sodium bicarbonate (NaHCO3) supplementation: one highly susceptible to the antistaphylococcal β-lactams oxacillin and cefazolin (NaHCO3 responsive) and one resistant to such agents (NaHCO3 nonresponsive). These phenotypes accurately predicted clearance profiles of MRSA from target tissues in experimental MRSA IE treated with each β-lactam. Mechanistically, NaHCO3 reduced the expression of two key genes involved in the MRSA phenotype, mecA and sarA, leading to decreased production of penicillin-binding protein 2a (that mediates methicillin resistance), in NaHCO3-responsive (but not in NaHCO3-nonresponsive) strains. Moreover, both cefazolin and oxacillin synergistically killed NaHCO3-responsive strains in the presence of the host defense antimicrobial peptide (LL-37) in NaHCO3-supplemented media. These findings suggest that AST of MRSA strains in NaHCO3-containing media may potentially identify infections caused by NaHCO3-responsive strains that are appropriate for β-lactam therapy

Scope and Predictive Genetic/Phenotypic Signatures of Bicarbonate (NaHCO3) Responsiveness and β-Lactam Sensitization in Methicillin-Resistant Staphylococcus aureus.

Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic β-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO3 responsiveness for OXA

A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA).

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed "bicarbonate (NaHCO3)-responsiveness" was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro "susceptibility" to standard β-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these β-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller-Hinton Broth (MHB); and 15 MIC-resistant to both β-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains

Ciprofioxacin in experimental Pseudomonas aeruginosa meningitis in rabbits

The potential of ciprofloxacin for the therapy of Pseudomonas aeruginosa meningitis was evaluated in an animal model by determining the penetration of the drug into CSF, its concentration-dependent killing characteristics in vivo, and its relative efficacy compared with ceftazidime and tobramycin. Meningitis was produced in 40 rabbits by intracisternal injection of 3 x 10 7 organisms. The drugs were administered intravenously over seven hours, and simultaneous serum and CSF samples were taken at 0, 1, 3, 5, and 7 h for determination of drug concentration and CSF bacterial counts. The percentage penetration of ciprofloxacin (18-4± 12'3; mean±standard deviation) in infected rabbits was substantially increased over that found in uninfected rabbits (4'1 ± 1· 3). The rate of bacterial killing for animals treated with ceftazidime (100 mg/kg/h) and high doses of tobramycin (2·5 mg/kg/h) was -0·51 ±0·13 (lOglO cfu/ml/h). This was similar to the rate of killing (-0-48±0·2) found when ciprofloxacin was infused at 5 mg/kg/h, a dose that produced a mean serum level of 6·7 ± 4·6 mg/I, which corresponds to concentrations achievable in humans. As dosages were increased (15 and 30 mg/kg/h), the rate of bacterial killing also increased (-0'70 ±O'I and -0·89±0·4 respectively; r = 0·7407; P<O·OI). The drug shows promise in the treatment of pseudomonas meningitis

Genetic Diversity of Arginine Catabolic Mobile Element in Staphylococcus epidermidis

BACKGROUND:The methicillin-resistant Staphylococcus aureus clone USA300 contains a novel mobile genetic element, arginine catabolic mobile element (ACME), that contributes to its enhanced capacity to grow and survive within the host. Although ACME appears to have been transferred into USA300 from S. epidermidis, the genetic diversity of ACME in the latter species remains poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS:To assess the prevalence and genetic diversity of ACME, 127 geographically diverse S. epidermidis isolates representing 86 different multilocus sequence types (STs) were characterized. ACME was found in 51% (65/127) of S. epidermidis isolates. The vast majority (57/65) of ACME-containing isolates belonged to the predominant S. epidermidis clonal complex CC2. ACME was often found in association with different allotypes of staphylococcal chromosome cassette mec (SCCmec) which also encodes the recombinase function that facilities mobilization ACME from the S. epidermidis chromosome. Restriction fragment length polymorphism, PCR scanning and DNA sequencing allowed for identification of 39 distinct ACME genetic variants that differ from one another in gene content, thereby revealing a hitherto uncharacterized genetic diversity within ACME. All but one ACME variants were represented by a single S. epidermidis isolate; the singular variant, termed ACME-I.02, was found in 27 isolates, all of which belonged to the CC2 lineage. An evolutionary model constructed based on the eBURST algorithm revealed that ACME-I.02 was acquired at least on 15 different occasions by strains belonging to the CC2 lineage. CONCLUSIONS/SIGNIFICANCE:ACME-I.02 in diverse S. epidermidis isolates were nearly identical in sequence to the prototypical ACME found in USA300 MRSA clone, providing further evidence for the interspecies transfer of ACME from S. epidermidis into USA300

Intermediate Vancomycin Susceptibility in a Community-associated MRSA Clone

We describe a case of treatment failure caused by a strain of USA300 community-associated methicillin-resistant Staphylococcus aureus (MRSA) with intermediate susceptibility to vancomycin and reduced susceptibility to daptomycin. The strain was isolated from the bone of a 56-year-old man with lumbar osteomyelitis after a 6-week treatment course of vancomycin for catheter-associated septic thrombophlebitis

Finding consensus for hamstring surgery in ambulatory children with cerebral palsy using the Delphi method

PURPOSE There is marked variation in indications and techniques for hamstring surgery in children with cerebral palsy. There is particular uncertainty regarding the indications for hamstring transfer compared to traditional hamstring lengthening. The purpose of this study was for an international panel of experts to use the Delphi method to establish consensus indications for hamstring surgery in ambulatory children with cerebral palsy. METHODS The panel used a five-level Likert-type scale to record agreement or disagreement with statements regarding hamstring surgery, including surgical indications and techniques, post-operative care, and outcome measures. Consensus was defined as at least 80% of responses being in the highest or lowest two of the five Likert-type ratings. General agreement was defined as 60%-79% falling into the highest or lowest two ratings. There was no agreement if neither of these thresholds was reached. RESULTS The panel reached consensus or general agreement for 38 (84%) of 45 statements regarding hamstring surgery. The panel noted the importance of assessing pelvic tilt during gait when considering hamstring surgery, and also that lateral hamstring lengthening is rarely needed, particularly at the index surgery. They noted that repeat hamstring lengthening often has poor outcomes. The panel was divided regarding hamstring transfer surgery, with only half performing such surgery. CONCLUSION The results of this study can help pediatric orthopedic surgeons optimize decision-making in their choice and practice of hamstring surgery for ambulatory children with cerebral palsy. This has the potential to reduce practice variation and significantly improve outcomes for ambulatory children with cerebral palsy. LEVEL OF EVIDENCE level V

Anterior distal femoral hemiepiphysiodesis in children with cerebral palsy: Establishing surgical indications and techniques using the modified Delphi method and literature review

PURPOSE The purpose of this study was to develop consensus for the surgical indications of anterior distal femur hemiepiphysiodesis in children with cerebral palsy using expert surgeon opinion through a modified Delphi technique. METHODS The panel used a 5-level Likert-type scale to record agreement or disagreement with 27 statements regarding anterior distal femur hemiepiphysiodesis. Consensus was defined as at least 80% of responses being in the highest or lowest 2 of the Likert-type ratings. General agreement was defined as 60%-79% falling into the highest or lowest 2 ratings. RESULTS For anterior distal femur hemiepiphysiodesis, 27 statements were surveyed: consensus or general agreement among the panelists was achieved for 22 of 27 statements (22/27, 82%) and 5 statements had no agreement (5/27, 18%). There was general consensus that anterior distal femur hemiepiphysiodesis is indicated for ambulatory children with cerebral palsy, with at least 2 years growth remaining, and smaller (<30 degrees) knee flexion contractures and for minimally ambulatory children to aid in standing/transfers. Consensus was achieved regarding the importance of close radiographic follow-up after screw insertion to identify or prevent secondary deformity. There was general agreement that percutaneous screws are preferred over anterior plates due to the pain and irritation associated with plates. Finally, it was agreed that anterior distal femur hemiepiphysiodesis was not indicated in the absence of a knee flexion contracture. CONCLUSION Anterior distal femur hemiepiphysiodesis can be used to treat fixed knee flexion contractures in the setting of crouch gait, but other associated lever arm dysfunctions must be addressed by single-event multilevel surgery. LEVEL OF EVIDENCE V

A photochemical model for the carbon-rich planet WASP-12b

The hot Jupiter WASP-12b is a heavily irradiated exoplanet in a short period orbit around a G0-star with twice the metallicity of the Sun. A recent thermochemical equilibrium analysis based on Spitzer and ground-based infrared observations suggests that the presence of $\ch4$ in its atmosphere and the lack of \h2o features can only be explained if the carbon-to-oxygen ratio in the planet's atmosphere is much greater than the solar ratio (\ctoo = 0.54). Here, we use a 1-D photochemical model to study the effect of disequilibrium chemistry on the observed abundances of \h2o, \com, \co2 and $\ch4$ in the WASP-12b atmosphere. We consider two cases: one with solar \ctoo and another with \ctoo = 1.08. The solar case predicts that \h2o and \com are more abundant than \co2 and $\ch4$, as expected, whereas the high \ctoo model shows that \com, C$_{2}$H$_{2}$ and HCN are more abundant. This indicates that the extra carbon from the high \ctoo model is in hydrocarbon species. \h2o photolysis is the dominant disequilibrium mechanism that alters the chemistry at higher altitudes in the solar \ctoo case, whereas photodissociation of C$_{2}$H$_{2}$ and HCN is significant in the super-solar case. Furthermore, our analysis indicates that $\c2h2$ is the major absorber in the atmosphere of WASP-12b and the absorption features detected near 1.6 and 8 micron may be arising from C$_{2}$H$_{2}$ rather than $\ch4$. The Hubble Space Telescope's WFC3 can resolve this discrepancy, as $\c2h2$ has absorption between $1.51 - 1.54$ microns, while $\ch4$ does not.Comment: Accepted to Astrophysical Journal. Reaction list available with the journal version of the pape