Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of ß-lactams, making them intractable to treatment. Although ß-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by mecA), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum ß-lactam resistance can arise following serial passaging of a mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 promoter, both of which play an instrumental role in ß-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant ß-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation ß-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the Km value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

High-Level Resistance of Staphylococcus aureus to ß-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4).

Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus, has been implicated in low-level resistance to ß-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to ß-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus These mutations did not appreciably alter the ß-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to ß-lactam antibiotics, such as ceftobiprole and ceftaroline.

Daptomycin-ß-Lactam Combinations in a Rabbit Model of Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Endocarditis.

Beta-lactams enhance the in vitro activity of daptomycin against methicillin-resistant strains of Staphylococcus aureus Experiments were performed in a rabbit model of aortic valve endocarditis caused by methicillin-resistant daptomycin-nonsusceptible S. aureus strain CB5054 to determine if a cephalosporin, ceftriaxone, administered as a once-daily dose of 100 mg/kg of body weight, or a carbapenem, ertapenem, administered as a once-daily dose of 40 mg/kg, improved the efficacy of daptomycin, administered as a once-daily dose of 12 mg/kg. Daptomycin was ineffective alone in reducing organism densities compared to untreated controls in vegetations and spleen, but densities were 1.4 log10 CFU/g lower in kidney. The combination of daptomycin plus ceftriaxone or daptomycin plus ertapenem reduced bacterial densities in all tissues compared to single agents, with 0.6 to 1.0 log10 CFU/g fewer organisms in vegetations, 1.5 to 2.5 log10 CFU/g fewer organisms in spleen, and 1.8 to 2.5 log10 CFU/g fewer organisms in kidney, although differences were statistically significant only in spleen for daptomycin plus ceftriaxone and in kidney for daptomycin plus ertapenem. Drug exposures in rabbits were less than those achievable in humans, which may have limited the in vivo activity, particularly in vegetations.

PBP 4 Mediates High-Level Resistance to New-Generation Cephalosporins in Staphylococcus aureus.

Staphylococcus aureus is an important cause of both hospital- and community-associated methicillin-resistant S. aureus (MRSA) infections worldwide. ß-Lactam antibiotics are the drugs of choice to treat S. aureus infections, but resistance to these and other antibiotics make treatment problematic. High-level ß-lactam resistance of S. aureus has always been attributed to the horizontally acquired penicillin binding protein 2a (PBP 2a) encoded by the mecA gene. Here, we show that S. aureus can also express high-level resistance to ß-lactams, including new-generation broad-spectrum cephalosporins that are active against methicillin-resistant strains, through an uncanonical core genome-encoded penicillin binding protein, PBP 4, a nonessential enzyme previously considered not to be important for staphylococcal ß-lactam resistance. Our results show that PBP 4 can mediate high-level resistance to ß-lactams.

Effects of selective and nonselective nonsteroidal anti-inflammatory drugs on antibiotic efficacy of experimental group A streptococcal myonecrosis.

BACKGROUND: Epidemiologic evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) contribute to more severe group A streptococcal (GAS) infections, yet a beneficial role for NSAIDs has been demonstrated in other experimental bacterial infections. METHODS: Nonselective (ketorolac tromethamine, ibuprofen, indomethacin), COX-1-selective (SC-560), or COX-2-selective (SC-236) NSAIDs ± antibiotics (penicillin, clindamycin) were given to mice challenged intramuscularly with M-type 3 GAS and disease course was followed for 14 days. RESULTS. All nonselective NSAIDs significantly accelerated mortality and reduced antibiotic efficacy; COX-selective NSAIDs had no significant effects. CONCLUSIONS: Use of nonselective NSAIDs, either alone or as adjuncts to antibiotic therapy, for GAS soft tissue infection may contribute to worse outcomes.

Pregnancy-related group a streptococcal infections: temporal relationships between bacterial acquisition, infection onset, clinical findings, and outcome.

Puerperal sepsis caused by group A Streptococcus (GAS) remains an important cause of maternal and infant mortality worldwide, including countries with modern antibiotic regimens, intensive care measures and infection control practices. To provide insights into the genesis of modern GAS puerperal sepsis, we reviewed the published cases and case series from 1974 to 2009, specifically seeking relationships between the likely source of pathogen acquisition, clinical signs, and symptoms at infection onset and patient outcomes that could provide clues for early diagnosis. Results suggest that the pathogenesis of pregnancy-related GAS infections in modern times is complex and not simply the result of exposure to GAS in the hospital setting. Additional research is needed to further explore the source of GAS, the specific M types involved, and the pathogenesis of these pregnancy-related infections to generate novel preventative and therapeutic strategies.

Do cardiomyocytes mount an immune response to Group A Streptococcus?

Some patients with Group A Streptococcal toxic shock syndrome (StrepTSS) develop a unique form of cardiomyopathy characterized by global hypokinesia and reduced cardiac index. Here we investigated the immune responses of cardiomyocytes to Group A Streptococcus both in vivo and in vitro. Our data demonstrate that cardiomyocyte-derived cytokines are produced following both direct GAS stimulation and after exposure to GAS-activated inflammatory cells. These locally produced, cardiomyocyte-derived cytokines may mediate cardiac contractile dysfunction observed in patients with StrepTSS-associated cardiomyopathy and may hold the key to our ability to attenuate this severe complication.

Augmented production of Panton-Valentine leukocidin toxin in methicillin-resistant and methicillin-susceptible Staphylococcus aureus is associated with worse outcome in a murine skin infection model.

The role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus infections is controversial. We used a mouse model of skin infection to compare the virulence of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains with different levels of PVL production. Differences in PVL production were not associated with mutations in the genes lukS-PV and lukF-PV. However, MSSA and MRSA strains that produced high levels of PVL caused larger skin abscesses, higher bacterial burdens, and more tissue inflammation than did low-PVL-producing strains. Together, these data suggest that (1) the effect of PVL on the pathogenesis of staphylococcal infection may depend on the level of toxin produced and (2) many strains of MSSA that cause soft-tissue infections produce higher levels of PVL than do MRSA strains.

In vitro production of panton-valentine leukocidin among strains of methicillin-resistant Staphylococcus aureus causing diverse infections.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus strains have recently been associated with severe necrotizing infections. Greater than 75% of these strains carry the genes for Panton-Valentine leukocidin (PVL), suggesting that this toxin may mediate these severe infections. However, to date, studies have not provided evidence of toxin production. METHODS: Twenty-nine community-acquired methicillin-resistant Staphylococcus aureus and 2 community-acquired methicillin-susceptible S. aureus strains were collected from patients with infections of varying severity. Strains were analyzed for the presence of lukF-PV and SCCmecA type. PVL production in lukF-PV gene-positive strains was measured by ELISA, and the amount produced was analyzed relative to severity of infection. RESULTS: Only 2 of the 31 strains tested, 1 methicillin-resistant Staphylococcus aureus abscess isolate and 1 nasal carriage methicillin-susceptible S. aureus isolate, were lukF-PV negative. All methicillin-resistant Staphylococcus aureus strains were SCCmec type IV. PVL was produced by all strains harboring lukF-PV, although a marked strain-to-strain variation was observed. Twenty-six (90%) of 29 strains produced 50-350 ng/mL of PVL; the remaining strains produced PVL in excess of 500 ng/mL. The quantity of PVL produced in vitro did not correlate with severity of infection. CONCLUSIONS: Although PVL likely plays an important role in the pathogenesis of these infections, its mere presence is not solely responsible for the increased severity. Factors that up-regulate toxin synthesis in vivo could contribute to more-severe disease and worse outcomes in patients with community-acquired methicillin-resistant Staphylococcus aureus infection.

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.

Balloon valvuloplasty for palliative treatment of tricuspid stenosis with right-to-left atrial-level shunting in a dog.

A 3.75-year-old castrated male Chesapeake Bay Retriever was referred for evaluation of tachypnea, exercise intolerance, and cyanosis. Echocardiographically, there was severe tricuspid stenosis and right-to-left atrial-level shunting of blood. Marked compensatory polycythemia had developed; the PCV was 75%. Balloon dilation of the tricuspid stenosis was performed. Subsequent echocardiographic examinations demonstrated a reduction in the pressure gradient across the tricuspid valve. The PCV returned to the reference range, and the dog's clinical status improved during the 12 months after the procedure. Tricuspid stenosis is an uncommon lesion in dogs and, in the dog of this report, was assumed to have resulted from tricuspid dysplasia. Cyanosis was a result of right-to-left shunting of blood. Limited treatment is available for dogs with cyanotic heart disease. In this dog, balloon dilation of the stenotic tricuspid valve was palliative.

Fatal S. aureus hemorrhagic pneumonia: genetic analysis of a unique clinical isolate producing both PVL and TSST-1.

In 2008, an unusual strain of methicillin-sensitive Staphylococcus aureus (MSSA68111), producing both Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1), was isolated from a fatal case of necrotizing pneumonia. Because PVL/TSST-1 co-production in S. aureus is rare, we characterized the molecular organization of these toxin genes in strain 68111. MSSA68111 carries the PVL genes within a novel temperate prophage we call ФPVLv68111 that is most similar, though not identical, to phage ФPVL--a phage type that is relatively rare worldwide. The TSST-1 gene (tst) in MSSA68111 is carried on a unique staphylococcal pathogenicity island (SaPI) we call SaPI68111. Features of SaPI68111 suggest it likely arose through multiple major recombination events with other known SaPIs. Both ФPVLv68111 and SaPI68111 are fully mobilizable and therefore transmissible to other strains. Taken together, these findings suggest that hypervirulent S. aureus have the potential to emerge worldwide.

Muscle injury, vimentin expression, and nonsteroidal anti-inflammatory drugs predispose to cryptic group A streptococcal necrotizing infection.

BACKGROUND: Myonecrosis due to group A streptococci (GAS) often develops at sites of nonpenetrating muscle injury, and nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the severity of these cryptic infections. We have previously shown that GAS bind to vimentin on injured skeletal muscles in vitro. The present study investigated whether vimentin up-regulation in injured muscles in vivo is associated with homing of circulating GAS to the injured site and whether NSAIDs facilitate this process. METHODS: M type 3 GAS were delivered intravenously 48 h after eccentric contraction (EC)-induced injury of murine hind-limb muscles. Vimentin gene expression and homing of GAS were followed by real-time reverse-transcriptase polymerase chain reaction and quantitative bacteriology of muscle homogenates, respectively. In separate experiments, ketorolac tromethamine (Toradol) was given 1 h before GAS infusion. RESULTS: Vimentin was up-regulated approximately 8-fold 48 h after EC. Significantly more GAS were found in moderately injured muscles than in noninjured controls. NSAIDs greatly augmented the number of GAS in injured muscles. CONCLUSIONS: Vimentin may tether circulating GAS to injured muscle, and NSAIDs enhance this process. Strategies targeting the vimentin-GAS interaction may prevent or attenuate GAS myonecrosis. Use of NSAIDs should increase suspicion of cryptic GAS infection in patients with increasing pain at sites of nonpenetrating muscle injury.

Evaluation of analgesia provided by the administration of epidural ketamine in dogs with a chemically induced synovitis.

OBJECTIVE: To determine if epidural ketamine provides analgesia in dogs with a chemically induced synovitis. STUDY DESIGN: Prospective randomized experimental trial. ANIMALS: Thirty-two healthy, adult mongrel dogs (13-30 kg). METHODS: (Part I) Synovitis was induced in the right stifle of 16 dogs and allowed to develop for 12 hours. Epidural injection at the lumbosacral space of either ketamine (2 mg kg(-1); n = 8) or placebo (n = 8) was performed. Limb use and pain were measured using a force platform and numerical rating scale (NRS). Assessments were performed before and at 12, 14, 16, 18, 20, and 24 hours after the induction of synovitis. (Part II) Epidural injection of either ketamine (n = 8) or placebo (n = 8) was performed immediately before the induction of synovitis. Analgesia was assessed as in Part I. Assessments occurred before and at 2, 4, 6, 8, and 12 hours after the induction of synovitis. RESULTS: (Part I) Vertical ground reaction forces (VGRF) significantly decreased and NRS scores of total pain significantly increased after the induction of synovitis in all dogs (p < 0.05). No significant differences in VGRF or NRS scores were measured between treatment groups at any assessment period. (Part II) Dogs that received ketamine had significantly lower NRS scores 2 hours after treatment (p < 0.05). NRS scores did not differ between groups at any other evaluation. VGRF did not differ significantly between treatment groups at any assessment period. CONCLUSION: Epidural ketamine at a dose of 2 mg kg(-1) administered after the development of synovitis does not provide significant levels of analgesia. Administration of ketamine before the induction of synovitis significantly decreased the NRS score 2 hours post-induction. CLINICAL RELEVANCE: Administration of epidural ketamine before tissue injury may provide analgesia of short duration in dogs.

Laparoscopic ovariohysterectomy in nine dogs.

Minimally invasive surgery has been found in humans to reduce pain, incidence of infections, and duration of hospitalization. Minimally invasive procedures are also being described in veterinary medicine. Laparoscopic ovariohysterectomy (OHE) was performed on nine, healthy, intact female dogs using a Harmonic scalpel. Creatine kinase values were determined both before and 12 hours following the laparoscopic OHE; the magnitude of the difference between preoperative and postoperative creatine kinase values did not correlate with length of operative time, length of incisions, or amount of hemorrhage. Complications included one dog that had an omental herniation that was primarily repaired and one dog with seroma formation. Median surgical time for all dogs was 60 minutes (range, 35 to 100 minutes).