PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

Integrating rapid diagnostics and antimicrobial stewardship improves outcomes in patients with antibiotic-resistant Gram-negative bacteremia.

BACKGROUND: An intervention for Gram-negative bloodstream infections that integrated mass spectrometry technology for rapid diagnosis with antimicrobial stewardship oversight significantly improved patient outcomes and reduced hospital costs. As antibiotic resistance rates continue to grow at an alarming speed, the current study was undertaken to assess the impact of this intervention in a challenging patient population with bloodstream infections caused by antibiotic-resistant Gram-negative bacteria. METHODS: A total of 153 patients with antibiotic-resistant Gram-negative bacteremia hospitalized prior to the study intervention were compared to 112 patients treated post-implementation. Outcomes assessed included time to optimal antibiotic therapy, time to active treatment when inactive, hospital and intensive care unit length of stay, all-cause 30-day mortality, and total hospital expenditures. RESULTS: Integrating rapid diagnostics with antimicrobial stewardship improved time to optimal antibiotic therapy (80.9 h in the pre-intervention period versus 23.2 h in the intervention period, P < 0.001) and effective antibiotic therapy (89.7 h versus 32 h, P < 0.001). Patients in the pre-intervention period had increased duration of hospitalization compared to those in the intervention period (23.3 days versus 15.3 days, P = 0.0001) and longer intensive care unit length of stay (16 days versus 10.7 days, P = 0.008). Mortality among patients during the intervention period was lower (21% versus 8.9%, P = 0.01) and our study intervention remained a significant predictor of survival (OR, 0.3; 95% confidence interval [CI], 0.12-0.79) after multivariate logistic regression. Mean hospital costs for each inpatient survivor were reduced $26,298 in the intervention cohort resulting in an estimated annual cost savings of $2.4 million (P = 0.002). CONCLUSIONS: Integration of rapid identification and susceptibility techniques with antimicrobial stewardship resulted in significant improvements in clinical and financial outcomes for patients with bloodstream infections caused by antibiotic-resistant Gram-negatives. The intervention decreased hospital and intensive care unit length of stay, total hospital costs, and reduced all-cause 30-day mortality.

Integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs.

CONTEXT: Early diagnosis of gram-negative bloodstream infections, prompt identification of the infecting organism, and appropriate antibiotic therapy improve patient care outcomes and decrease health care expenditures. In an era of increasing antimicrobial resistance, methods to acquire and rapidly translate critical results into timely therapies for gram-negative bloodstream infections are needed. OBJECTIVE: To determine whether mass spectrometry technology coupled with antimicrobial stewardship provides a substantially improved alternative to conventional laboratory methods. DESIGN: An evidence-based intervention that integrated matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, rapid antimicrobial susceptibility testing, and near-real-time antimicrobial stewardship practices was implemented. Outcomes in patients hospitalized prior to initiation of the study intervention were compared to those in patients treated after implementation. Differences in length of hospitalization and hospital costs were assessed in survivors. RESULTS: The mean hospital length of stay in the preintervention group survivors (n = 100) was 11.9 versus 9.3 days in the intervention group (n = 101; P = .01). After multivariate analysis, factors independently associated with decreased length of hospitalization included the intervention (hazard ratio, 1.38; 95% confidence interval, 1.01-1.88) and active therapy at 48 hours (hazard ratio, 2.9; confidence interval, 1.15-7.33). Mean hospital costs per patient were $45 709 in the preintervention group and $26 162 in the intervention group (P = .009). CONCLUSIONS: Integration of rapid identification and susceptibility techniques with antimicrobial stewardship significantly improved time to optimal therapy, and it decreased hospital length of stay and total costs. This innovative strategy has ramifications for other areas of patient care.

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice.

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

Epidemiology of pediatric tuberculosis using traditional and molecular techniques: Houston, Texas.

OBJECTIVE: To investigate the transmission dynamics of pediatric tuberculosis (TB) by analyzing the clinical characteristics with the molecular profiles of Mycobacterium tuberculosis isolates during a 5-year period. METHODS: A retrospective review of a prospective population-based active surveillance and molecular epidemiology project was conducted in private and public pediatric clinics within Houston and Harris County, Texas. The study population consisted of patients who had pediatric TB diagnosed from October 1, 1995, through September 30, 2000. Cases and potential source cases (PSC) were interviewed using a standardized questionnaire. Available Mycobacterium tuberculosis isolates from cases and PSCs were characterized and compared by IS6110 restriction fragment length polymorphism, spoligotyping, and genetic group assignment. Clinical characteristics were described, and molecular characterizations were compared. Data were analyzed by using EpiInfo 6.02b and SAS 8.2. RESULTS: A total of 220 (92%) of 238 pediatric TB cases were included. Epidemiologic and clinical findings were consistent with previous studies. Molecular profiles from 3 cases did not match the profile of PSC. Four previously unknown PSCs were identified using molecular techniques. Fifty-one (71.8%) of 71 isolates matched at least 1 other Houston Tuberculosis Initiative TB database isolate and were grouped into 33 molecular clusters. Cases were more likely to be clustered when the patients were younger than 5 years, identified a source case, or were US born. CONCLUSIONS: Traditional contact tracing may not always be accurate, and molecular characterization can lead to identification of previously unrecognized source cases. Recent transmission plays a significant role in the transmission of TB to children as evident by the high degree of clustering found in our study population.

Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity.

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus delta-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of delta-toxin deformylation. We observed that during exponential and early postexponential growth, delta-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated delta-toxin accumulated after the early postexponential phase. The transition from producing both species of delta-toxin to producing only formyl-methionine-containing delta-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of delta-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that delta-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated delta-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated delta-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.

Identification and characterization of HtsA, a second heme-binding protein made by Streptococcus pyogenes.

Group A streptococci (GAS) can use heme and hemoproteins as sources of iron. However, the machinery for heme acquisition in GAS has not been firmly revealed. Recently, we identified a novel heme-associated cell surface protein (Shp) made by GAS. The shp gene is cotranscribed with eight downstream genes, including spy1795, spy1794, and spy1793 encoding a putative ABC transporter (designated HtsABC). In this study, spy1795 (designated htsA) was cloned from a serotype M1 strain, and recombinant HtsA was overexpressed in Escherichia coli and purified to homogeneity. HtsA binds 1 heme molecule per molecule of protein. HtsA was produced in vitro and localized to the bacterial cell surface. GAS up-regulated transcription of htsA in human blood compared with that in Todd-Hewitt broth supplemented with 0.2% yeast extract. The level of the htsA transcript dramatically increased under metal cation-restricted conditions compared with that under metal cation-replete conditions. The cation content, cell surface location, and gene transcription of HtsA were also compared with those of MtsA and Spy0385, the lipoprotein components of two other putative iron acquisition ABC transporters of GAS. Our results suggest that HtsABC is an ABC transporter that may participate in heme acquisition in GAS.