Joan Pawlak

Broad-Range Bacterial Polymerase Chain Reaction for Early Detection of Bacterial Meningitis

The diagnosis of bacterial meningitis often depends on isolation of bacteria on culture, which may take 24-48 h. DNA amplification techniques could provide rapid diagnosis, which would guide the clinician in antimicrobial therapy decisions. This study determined the clinical utility of polymerase chain reaction (PCR) for the diagnosis of meningitis with use of a broad range of bacterial primers. Seventy-four cerebrospinal fluid specimens obtained from 70 patients were subjected to PCR with use of primers derived from conserved regions of the bacterial 16S RNA gene. The test characteristics for the broad-range bacterial PCR were as follows: sensitivity, 100%; specificity, 98.2%; positive predictive value, 94.4%; and negative predictive value, 100%. Broad-range bacterial PCR may be useful for excluding the diagnosis of meningitis, and the results may influence the decision to initiate or discontinue antimicrobial therapy.

In Vitro Activity of Ceftaroline against Community-Associated Methicillin-Resistant, Vancomycin-Intermediate, Vancomycin-Resistant, and Daptomycin-Nonsusceptible Staphylococcus aureus Isolates

ABSTRACT This study assessed the in vitro activities of ceftaroline and five comparator agents against a collection of Staphylococcus aureus isolates. Ceftaroline demonstrated potent activity against community-associated methicillin-resistant S. aureus (CA-MRSA) isolates and showed bactericidal activity against vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates. Ceftaroline may represent a bactericidal treatment option for infections caused by these pathogens.

Changing epidemiology of community-onset methicillin-resistant Staphylococcus aureus bacteremia

OBJECTIVES To review cases of community-onset Staphylococcus aureus bacteremia and to evaluate whether the risk factors and epidemiology of methicillin-resistant S. aureus (MRSA) bacteremia have changed from early reports. DESIGN Retrospective case-comparison study of community-onset MRSA (n = 26) and methicillin-susceptible S. aureus (MSSA) (n = 26) bacteremias at our institution. SETTING A 600-bed urban academic medical center. PATIENTS Twenty-six patients with community-onset MRSA bacteremia were compared with 26 patients with community-onset MSSA bacteremia. Molecular analysis was performed on S. aureus isolates from the 26 MRSA cases as well as from 13 cases of community-onset S. aureus bacteremia from 1980 and 9 cases of nosocomial S. aureus bacteremia from 2001. RESULTS The two groups were similar except that patients with MRSA bacteremia were more likely to have presented from a long-term-care facility (26.9% vs 4%; P = .05) and to have had multiple admissions within the preceding year (46% vs 15%; P = .03). Clamped homogeneous electric fields analysis of MRSA isolates from 1982 revealed predominantly that one clone was the epidemic strain, whereas there were 14 unique strains among current community-onset isolates. Among current nosocomial isolates, 3 patterns were identified, all of which were present in the community-onset cases. CONCLUSIONS Previously described risk factors for MRSA acquisition may not be helpful in predicting disease due to the polyclonal spread of MRSA in the community. Unlike early outbreaks of MRSA in patients presenting from the community, current acquisition appears to be polyclonal and is usually related to contact with the healthcare system.

In Vitro Susceptibilities and Molecular Analysis of Vancomycin-Intermediate and Vancomycin-Resistant Staphylococcus aureus Isolates

BACKGROUND There is increasing frequency of vancomycin-intermediate and -resistant Staphylococcus aureus (VISA and VRSA) isolates identified in clinical practice. There are limited reports evaluating susceptibility patterns and molecular characteristics of these strains. METHODS Laboratory analysis was performed on 13 VRSA and 33 VISA isolates, including susceptibility testing by broth microdilution, detection of Panton-Valentine leukocidin (PVL) genes, arginine catabolic mobile element (ACME), and staphylococcal cassette chromosome mec typing using polymerase chain reaction. Strain typing using pulsed-field gel electrophoresis (PFGE) was performed on VRSA isolates. RESULTS Telavancin, linezolid, tigecycline, and minocycline were active against >90% of VISA isolates, while >90% of VRSA isolates were susceptible to ceftaroline, daptomycin, linezolid, minocyline, tigecycline, rifampin, and trimethoprim/sulfamethoxazole. There were no VISA or VRSA isolates that carried PVL genes or ACME, and most strains (69.8%) were staphylococcal cassette chromosome mec type II. VRSA isolates were predominantly related to USA100 (53.8%) and none were related to USA300 or USA400. CONCLUSIONS A large number of available antimicrobial agents retain very good in vitro activity against VRSA and VISA isolates. The present isolates appear to be derived from healthcare-associated strains based on the absence of features associated with community-associated strains, and VRSA isolates are polyclonal by PFGE.

In vitro activity of oritavancin against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin-non-susceptible S. aureus (DNSSA)

Isolates of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA) are increasing in frequency and new antistaphylococcal therapies are needed. Microdilution testing using Mueller-Hinton broth was used to determine the minimal inhibitory concentrations (MICs) of oritavancin and nine additional antimicrobial agents against 92 CA-MRSA, 23 VISA, 7 DNSSA and 10 VRSA isolates. Minimal bactericidal concentrations were also determined. Pulsed-field gel electrophoresis (PFGE) was performed. Staphylococcal cassette chromosome mec (SCCmec) typing as well as assays for Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. Oritavancin was more bactericidal than any of the other comparators against CA-MRSA and demonstrated excellent activity against VRSA and VISA.

Rapid diagnosis of septic arthritis using 16S rDNA PCR: a comparison of 3 methods.

Few studies address the utility of molecular techniques for diagnosis of infection in synovial fluid (SF). We evaluated 3 different methods using 16S rDNA polymerase chain reaction (PCR) on 63 specimens for the diagnosis of joint infection. SF samples were classified as normal, inflammatory, or septic based on the patients clinical and laboratory results. Samples were analyzed by conventional PCR using primers for the bacterial 16S rDNA gene and by real-time PCR utilizing 2 different sets of primers for the target gene 16S rDNA. PCR results were compared to culture results. All inflammatory and normal SF samples were culture negative. There was concordance with 10 of the 16 septic samples by 2 of the PCR methods. When comparing 3 methods for rapid detection of septic arthritis, real-time PCR using SYBR-Green I and conventional PCR demonstrated favorable test characteristics, but need further study.

Community-Associated Methicillin-Resistant Staphylococcus aureus Causing Chronic Pneumonia

A young woman presented with pneumonia of a 3-month duration with predominantly nodular pulmonary infiltrates. Methicillin-resistant Staphylococcus aureus was identified in multiple cultures of sputum specimens. According to findings of pulsed-field gel electrophoresis, the isolate was identical to USA 300 and carried a type IV Staphylococcus cassette chromosome mec type IV gene and the genes for Panton-Valentine leukocidin.

Ceftaroline Heteroresistant Staphylococcus aureus

ABSTRACT Heteroresistance refers to the presence, within a large population of antimicrobial-susceptible microorganisms, of subpopulations with lesser susceptibilities. Ceftaroline is a novel cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to detect the prevalence of ceftaroline heteroresistance in vitro in a select group of S. aureus strains. There were 57 isolates selected for evaluation, 20 MRSA, 20 vancomycin-intermediate S. aureus (VISA), 7 daptomycin-nonsusceptible S. aureus (DNSSA), 6 linezolid-nonsusceptible S. aureus (LNSSA), and 4 heteroresistant VISA (hVISA) isolates. MICs and minimal bactericidal concentrations were determined using the broth microdilution method according to CLSI guidelines. All of the isolates were analyzed by pulsed-field gel electrophoresis. The staphylococcal cassette chromosome mec element (SCCmec) types were determined by a multiplex PCR. Population analysis profiles (PAPs) were performed to determine heteroresistance for all of the isolates using plates made by adding various amounts of ceftaroline to brain heart infusion agar. The frequencies of resistant subpopulations were 1 in 104 to 105 organisms. We determined that 12 of the 57 (21%) isolates tested were ceftaroline-heteroresistant S. aureus (CHSA). CHSA occurred among strains with reduced susceptibilities to vancomycin, daptomycin, and linezolid but occurred in none of the USA-300 isolates tested. Evaluation of the heteroresistant strains demonstrated that the phenotype was unstable. Further studies are needed to determine whether CHSA has a role in clinical failures and to determine the implications of our study findings.

In vitro Activity of Retapamulin against Staphylococcus aureus Resistant to Various Antimicrobial Agents

ABSTRACT Retapamulin and six other antimicrobial agents were evaluated against 155 methicillin-resistant Staphylococcus aureus (MRSA) isolates, including strains resistant to vancomycin, linezolid, daptomycin, and mupirocin by microdilution tests. Time-kill assays were performed against representative MRSA, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus (VRSA) isolates. Retapamulin and mupirocin demonstrated MIC90s of 0.12 μg/ml and 8 μg/ml, respectively, with resistance seen in 2.6% and 10% of isolates, respectively. Retapamulin maintained good activity against 94% (15/16) of mupirocin-resistant isolates.

VISA–Daptomycin non-susceptible Staphylococcus aureus frequently demonstrate non-susceptibility to Telavancin

Telavancin was evaluated against S. aureus isolates with reduced susceptibility to other antimicrobial agents using two broth microdilution methods and Etest® strips. The three methods provided comparable results. Differences in telavancin susceptibility versus non-susceptibility were noted mainly in the VISA-daptomycin non-susceptible group of isolates. In this group the percent susceptibility was 38% for the Etest® method and 50% and 54% for the 2 broth microdilution methods. All differences in susceptibility were within one 2-fold dilution.