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.

A genomic day in the life of a clinical microbiology laboratory.

Next-generation sequencing technology is available to many clinical laboratories; however, it is not yet widely used in routine microbiology practice. To demonstrate the feasibility of using whole-genome sequencing in a routine clinical microbiology workflow, we sequenced the genome of every organism isolated in our laboratory for 1 day.

Strategy for rapid identification and antibiotic susceptibility testing of gram-negative bacteria directly recovered from positive blood cultures using the Bruker MALDI Biotyper and the BD Phoenix system.

Decreasing the time to species identification and antibiotic susceptibility determination of strains recovered from patients with bacteremia significantly decreases morbidity and mortality. Herein, we validated a method to identify Gram-negative bacteria directly from positive blood culture medium using the Bruker MALDI Biotyper and to rapidly perform susceptibility testing using the BD Phoenix.

Extensively Drug-Resistant Myroides odoratus in Critically Ill Patients: A Case Series and Literature Review.

The bacterial genus Myroides, like other members of the Flavobacteriaceae family, consists of aerobic, non-motile, Gram-negative bacilli. Myroides spp. is considered predominantly opportunistic pathogens as, historically, most documented infections have been in immunocompromised individuals. Along with advancements in molecular assay testing, there are growing reports of clinically relevant Myroides spp. infections in immunocompetent individuals. These organisms display broad antimicrobial resistance, and while research into their mechanisms of resistance is progressing, genetic testing has revealed metallo-ß-lactamases present in their genome. The sporadic identification of Myroides spp. and ongoing clarification of resistance patterns make empiric treatment difficult. This report documents two cases of extensively drug-resistant Myroides odoratus isolated from critically ill but otherwise immunocompetent patients followed by a review of available literature on Myroides spp. antibiotic sensitivities. Our findings indicate that minocycline and moxifloxacin have the highest documented in vitro activity against Myroides spp.

Spontaneous bacterial pericarditis with tamponade due to Ureaplasma spp.

Infectious pericardial effusion with tamponade is an uncommon but life-threatening disease. We report an unusual case of spontaneous Ureaplasma pericardial effusion with tamponade associated with pneumonia, pleural effusion, and urinary tract infection. All published cases of clinically invasive Ureaplasma infections in the adult population are also reviewed.

Real-time Communication With Health Care Providers Through an Online Respiratory Pathogen Laboratory Report.

We implemented a real-time report to distribute respiratory pathogen data for our 8-hospital system to anyone with an Internet connection and a web browser. Real-time access to accurate regional laboratory observation data during an epidemic influenza season can guide diagnostic and therapeutic strategies.

Validation of the MycoAlign system for Mycobacterium spp. identification.

Validation of the MycoAlign assay, a newly developed Mycobacterium spp. identification system based on internal transcribed spacer-1 sequencing, was performed using 50 acid-fast bacilli (AFB)-positive clinical laboratory specimens. Forty-three (86%) diagnostic-level results were obtained, including 38 Mycobacterium spp. and 5 other AFB-positive genera. Three isolates (6%) had suboptimal identity scores with high probability (81-87% identity score). Four (8%) mixed-pattern results were obtained. Forty-five (90%) observations were concordant with the species identification by standard methods, including all controls.

Integrating Rapid Diagnostics and Antimicrobial Stewardship in Two Community Hospitals Improved Process Measures and Antibiotic Adjustment Time.

OBJECTIVE To assess the impact of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry for rapid pathogen identification directly from early-positive blood cultures coupled with an antimicrobial stewardship program (ASP) in two community hospitals. Process measures and outcomes prior and after implementation of MALDI-TOF/ASP were evaluated. DESIGN Multicenter retrospective study. SETTING Two community hospitals in a system setting, Houston Methodist (HM) Sugar Land Hospital (235 beds) or HM Willowbrook Hospital (241 beds). PATIENTS Patients ≥ 18 years of age with culture-proven Gram-negative bacteremia. INTERVENTION Blood cultures from both hospitals were sent to and processed at our central microbiology laboratory. Clinical pharmacists at respective hospitals were notified of pathogen ID and susceptibility results. RESULTS We evaluated 572 patients for possible inclusion. After pre-defined exclusion criteria, 151 patients were included in the pre-intervention group and 242 were included in the intervention group. After MALDI-TOF/ASP implementation, the mean identification time after culture positivity was significantly reduced from 32 hours (±16 hours) to 6.5 hours (±5.4 hours) (P<.001); mean time to susceptibility results was significantly reduced from 48 (±22) hours to 23 (±14) hours (P<.001); and time to therapy adjustment was significantly reduced from 75 (±59) hours to 30 (±30) hours (P<.001). Mean hospital costs per patient were $3,411 less in the intervention group compared with the pre-intervention group ($18,645 vs $15,234; P=.04). CONCLUSION This study is the first to analyze the impact of MALDI-TOF coupled with an ASP in a community hospital setting. Time to results significantly differed with the use of MALDI-TOF, and time to appropriate therapy was significantly improved with the addition of ASP.

Improving positive blood culture removal time significantly decreases total processing time.

CONTEXT: Timely processing of blood cultures with positive results, including Gram staining and notification of clinicians, is a critical function of the clinical microbiology laboratory. Analysis of processing time in our laboratory revealed opportunities to enhance workflow efficiency. We found that the average time from positive blood culture result to removal of the bottle for processing (positive-to-removal [PR] time) was inadequate for our rapid pathogen identification program. OBJECTIVE: To determine whether increased vigilance about PR time and prioritization of laboratory resources would decrease PR time and total processing time. DESIGN: We performed a retrospective analysis of blood culture PR time 7 months before and 7 months after an in-service meeting during which the importance of PR time was emphasized, and corrective measures were implemented. RESULTS: Before the in-service meeting, the average PR time for 5057 samples was 38 minutes, with an aggregate time of 192,251 minutes. Unexpectedly, we discovered that only 51.8% (2617 of 5057) of the positive blood cultures were removed in less than 10 minutes. After the in-service meeting, for 5293 samples, the average PR time improved to 8 minutes, the aggregate time improved to 44,630 minutes, and 84.5% (4470 of 5293) of the positive blood cultures were removed in less than 10 minutes. These improvements reduced the time to telephone notification of the Gram stain results to a caregiver by 46.7% (from 105 minutes to 56 minutes). CONCLUSIONS: Increased awareness of barriers to rapid pathogen identification and interventions for improving performance time significantly enhanced care of patients with bloodstream infections.

Fungal sinusitis: histologic spectrum and correlation with culture.

Fungi are important etiologic agents of sinusitis. However, features of fungal sinusitis including the histologic spectrum, diagnostic mishaps, incidence, and fungal types have not been systematically studied. From 1996 through 2001, a total of 788 surgical pathology sinus specimens from 384 cases was retrieved. Fungal sinusitis was diagnosed in 58 specimens (7%) from 47 cases (12%). Four histologic categories of fungal sinusitis were identified: (1) allergic fungal sinusitis in 34 cases (copious mucin, abundant eosinophils, Charcot-Leyden crystals (so-called allergic mucin), with rare noninvasive fungal hyphae); (2) mycetoma/fungus ball in 11 cases (tightly packed fungal hyphae without allergic mucin or tissue invasion); (3) chronic invasive fungal sinusitis in 1 case (tissue granulomas with fungal hyphae); and (4) acute fulminant fungal sinusitis in 1 case (fungal vascular invasion). The diagnosis was initially missed in 16/34 (47%) cases of allergic fungal sinusitis despite typical features; incorrect classification was noted in 47% of cases. Sixty-seven percent of cases had positive fungal cultures, dematiaceous fungi being the most common. Allergic fungal sinusitis accounted for the majority of fungal sinusitis. Although misdiagnosis or incorrect classification is rather frequent for fungal sinusitis, awareness of the distinctive morphologic features of this entity may prevent these errors.

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.

Rapidly progressive, fatal, inhalation anthrax-like infection in a human: case report, pathogen genome sequencing, pathology, and coordinated response.

CONTEXT: Ten years ago a bioterrorism event involving Bacillus anthracis spores captured the nation's interest, stimulated extensive new research on this pathogen, and heightened concern about illegitimate release of infectious agents. Sporadic reports have described rare, fulminant, and sometimes fatal cases of pneumonia in humans and nonhuman primates caused by strains of Bacillus cereus , a species closely related to Bacillus anthracis. OBJECTIVES: To describe and investigate a case of rapidly progressive, fatal, anthrax-like pneumonia and the overwhelming infection caused by a Bacillus species of uncertain provenance in a patient residing in rural Texas. DESIGN: We characterized the genome of the causative strain within days of its recovery from antemortem cultures using next-generation sequencing and performed immunohistochemistry on tissues obtained at autopsy with antibodies directed against virulence proteins of B anthracis and B cereus. RESULTS: We discovered that the infection was caused by a previously unknown strain of B cereus that was closely related to, but genetically distinct from, B anthracis . The strain contains a plasmid similar to pXO1, a genetic element encoding anthrax toxin and other known virulence factors. Immunohistochemistry demonstrated that several homologs of B anthracis virulence proteins were made in infected tissues, likely contributing to the patient's death. CONCLUSIONS: Rapid genome sequence analysis permitted us to genetically define this strain, rule out the likelihood of bioterrorism, and contribute effectively to the institutional response to this event. Our experience strongly reinforced the critical value of deploying a well-integrated, anatomic, clinical, and genomic strategy to respond rapidly to a potential emerging, infectious threat to public health.

Mycobacterial synovitis caused by slow-growing nonchromogenic species: eighteen cases and a review of the literature.

CONTEXT: Slow-growing nonchromogenic mycobacterial species are an infrequent cause of soft tissue infection. Because these organisms are rare, they are not often initially considered in the differential diagnosis of synovitis. OBJECTIVE: To evaluate the clinical and pathologic characteristics of patients with synovitis resulting from slow-growing nonchromogenic mycobacterial species. DESIGN: A 20-year retrospective review of records from The Methodist Hospital Microbiology Laboratory identified 18 culture-positive cases of synovitis that resulted from slow-growing nonchromogenic mycobacteria, including 14 caused by Mycobacterium avium complex, 1 caused by Mycobacterium malmoense, 1 caused by Mycobacterium haemophilum, and 2 caused by Mycobacterium nonchromogenicum isolates. In addition, a comprehensive literature search revealed an additional 48 cases of synovitis caused by slow-growing nonchromogenic mycobacteria. RESULTS: The historic literature described the majority of the 48 patients as previously healthy, elderly individuals with a several-month history of monoarticular pain and swelling in the small joints of the upper extremity. In contrast, the current series demonstrated the probable role of multiple chronic coexisting medical conditions in promoting disease susceptibility. These patients were also unique in their significantly younger age distribution and diversity of infection sites. Histologic examination and direct acid-fast bacteria stains generally did not aid the diagnosis. Amputation was performed in 2 patients because of delayed identification of disease. CONCLUSIONS: The current series demonstrates that difficult identification and infrequent occurrence cause these organisms to be overlooked by physicians and laboratory personnel. A heightened clinical suspicion for slow-growing nonchromogenic mycobacterial species is necessary when routine culture and histopathologic findings do not readily isolate an organism, or when the patient does not respond to antibiotic and anti-inflammatory treatment.