Janet Hindler

Increased Vancomycin MICs for Staphylococcus aureus Clinical Isolates from a University Hospital during a 5-Year Period

ABSTRACT Staphylococcus aureus is one of the most commonly isolated organisms in nosocomial infections. While the prevalence of methicillin-resistant S. aureus (MRSA) continues to increase worldwide, there is concern about an increase in vancomycin MICs among S. aureus strains. The prevalence of MRSA and vancomycin MIC trends in S. aureus from patients in a university hospital were analyzed. Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference broth microdilution MIC testing was performed on all clinically relevant S. aureus isolates from January 2000 through December 2004. A total of 6,003 S. aureus isolates were analyzed. No vancomycin-resistant S. aureus isolates were detected. One MRSA isolate had a vancomycin MIC of 8 μg/ml and was confirmed as vancomycin-intermediate S. aureus. Among the 6,002 remaining isolates, a shift in vancomycin MICs from ≤0.5 to 1.0 μg/ml was observed during the 5-year period. The percentage of S. aureus isolates with a vancomycin MIC of 1 μg/ml in 2004 was significantly higher than the percentage of isolates in 2000 (70.4% versus 19.9%; P < 0.01). This vancomycin MIC shift was more notable in methicillin-susceptible S. aureus. Our 5 years of routine testing of clinical isolates using the CLSI reference broth microdilution MIC method demonstrated a tendency toward decreasing susceptibility to vancomycin in S. aureus.

The emerging problem of linezolid-resistant Staphylococcus

The oxazolidinone antibiotic linezolid has demonstrated potent antimicrobial activity against Gram-positive bacterial pathogens, including methicillin-resistant staphylococci. This article systematically reviews the published literature for reports of linezolid-resistant Staphylococcus (LRS) infections to identify epidemiological, microbiological and clinical features for these infections. Linezolid remains active against >98% of Staphylococcus, with resistance identified in 0.05% of Staphylococcus aureus and 1.4% of coagulase-negative Staphylococcus (CoNS). In all reported cases, patients were treated with linezolid prior to isolation of LRS, with mean times of 20.0 ± 47.0 months for S. aureus and 11.0 ± 8.0 days for CoNS. The most common mechanisms for linezolid resistance were mutation (G2576T) to the 23S rRNA (63.5% of LRSA and 60.2% of LRCoNS) or the presence of a transmissible cfr ribosomal methyltransferase (54.5% of LRSA and 15.9% of LRCoNS). The emergence of linezolid resistance in Staphylococcus poses significant challenges to the clinical treatment of infections caused by these organisms, and in particular CoNS.

Molecular Correlation for the Treatment Outcomes in Bloodstream Infections Caused by Escherichia coli and Klebsiella pneumoniae with Reduced Susceptibility to Ceftazidime

Data are limited on outcomes of treatment with extended-spectrum cephalosporins (ESCs) for infections caused by Enterobacteriaceae that produce extended-spectrum beta-lactamases (ESBLs). This study describes the largest treatment experience of a nonoutbreak series of bloodstream infections caused by strains of Escherichia coli (23 episodes) and Klebsiella pneumoniae (13 episodes) with a ceftazidime minimal inhibitory concentration of > or =2 microg/mL. E. coli isolates produced a greater variety of beta-lactamase types than did K. pneumoniae isolates, among which ESBL production was predominant. Five ESBL types were identified: TEM-12, TEM-71, TEM-6, SHV-12, and SHV-5. Most patients were treated empirically with an ESC-based regimen. A favorable response to treatment with a nonceftazidime ESC was observed when the causative pathogen produced either TEM-6 or TEM-12; ceftazidime treatment was associated with failure of therapy in all patients. Despite the limited clinical success, ESCs are currently not recommended for the treatment of serious infections caused by ESBL-producing strains.

Multicenter Evaluation of BBL CHROMagar MRSA Medium for Direct Detection of Methicillin-Resistant Staphylococcus aureus from Surveillance Cultures of the Anterior Nares

ABSTRACT Active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) is among the strategies recommended by the Society for Healthcare Epidemiology of America for control of nosocomial MRSA infections. Infection control and laboratory personnel desire rapid, sensitive, and inexpensive methods to enhance surveillance activities. A multicenter study was performed to evaluate a new selective and differential chromogenic medium, BBL CHROMagar MRSA (C-MRSA) medium (BD Diagnostics, Sparks, MD), which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens taken from the anterior nares. Specimens were inoculated to C-MRSA and Trypticase soy agar with 5% sheep blood agar (TSA II, BD Diagnostics). Mauve colonies on C-MRSA at 24 h and 48 h and suspicious colonies on TSA II were confirmed as Staphylococcus aureus by Gram stain morphology and a coagulase test. In addition, the results of C-MRSA were compared to results of susceptibility testing (five different methods) of S. aureus strains isolated on TSA II. A total of 2,015 specimens were inoculated to C-MRSA and TSA II. Three hundred fifty-four S. aureus isolates were recovered; 208 (59%) were oxacillin (methicillin) susceptible and 146 (41%) were oxacillin resistant (MRSA). On C-MRSA, 139/146 or 95.2% of MRSA isolates were recovered, whereas recovery on TSA II was 86.9% (127/146) (P = 0.0027). The overall specificity of C-MRSA was 99.7%. When C-MRSA was compared to each susceptibility testing method, the sensitivity and specificity, respectively, were as follows: oxacillin MIC by broth microdilution, 94.4% and 96.7%; oxacillin screen agar, 94.3% and 96.7%; PBP2′ latex agglutination, 93.7% and 98.5%; cefoxitin disk diffusion, 95.0% and 98.1%; and mecA PCR, 95.1% and 98.1%. In this study, C-MRSA was superior to TSA II for recovery of MRSA from surveillance specimens obtained from the anterior nares and was comparable to conventional, rapid, and molecular susceptibility methods for the identification of MRSA isolates.

SME-Type Carbapenem-Hydrolyzing Class A β-Lactamases from Geographically Diverse Serratia marcescens Strains

ABSTRACT Three sets of carbapenem-resistant Serratia marcescensisolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two β-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 β-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U.S. isolates had β-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing β-lactamases first described in London has now been identified inS. marcescens isolates across the United States.

Quinupristin/Dalfopristin Therapy for Infections Due to Vancomycin-Resistant Enterococcus faecium

The efficacy and safety of quinupristin/dalfopristin for treatment of infections due to vancomycin-resistant Enterococcus faecium were evaluated in 24 hospitalized patients with documented infections (19 bacteremias, 5 localized infections) caused by vancomycin-resistant E. faecium that was susceptible to quinupristin/dalfopristin in vitro. Patients received iv quinupristin/dalfopristin at a dosage of either 7.5 mg/kg every 8 h or 5 mg/kg every 8 h. A favorable clinical response (cure or improvement) occurred in 19 (83%) of 23 evaluable patients; bacteriologic eradication occurred in 17 (74%) of 23 evaluable patients. A favorable clinical response was observed in 12 (80%) of 15 patients who were treated with 7.5 mg/kg of quinupristin/dalfopristin every 8 h and in 7 (88%) of 8 patients treated with 5 mg/kg of quinupristin/dalfopristin every 8 h. Two of four treatment failures were associated with a decrease in the in vitro susceptibility of vancomycin-resistant E. faecium to quinupristin/dalfopristin. Superinfections developed in 6 patients (26%), but only one was caused by Enterococcus faecalis that was resistant to quinupristin/dalfopristin. Myalgias and arthralgias were the only adverse events related to quinupristin/dalfopristin. These conditions occurred in 8 (33%) of 24 patients and were dose-related (8 cases in 16 patients treated with 7.5 mg/kg of quinupristin/dalfopristin every 8 h, no cases in 8 patients treated with 5 mg/kg every 8 h). Mortality associated with vancomycin-resistant E. faecium infection was 17% (4 of 23 patients), whereas mortality from other causes was 52% (12 of 23 patients). These results suggest that quinupristin/dalfopristin is effective as treatment for vancomycin-resistant E. faecium infections in critically ill patients with serious underlying conditions. Except for myalgias and arthralgias at higher dosages, the drug is well-tolerated.

Application of an rRNA Probe Matrix for Rapid Identification of Bacteria and Fungi from Routine Blood Cultures

ABSTRACT One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at −70°C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).

Selection of Strains for Quality Assessment of the Disk Induction Method for Detection of Inducible Clindamycin Resistance in Staphylococci: a CLSI Collaborative Study

ABSTRACT A nine-laboratory collaborative study was conducted to select positive and negative quality assessment control strains for the detection of inducible clindamycin resistance in staphylococci. Four strains of Staphylococcus aureus were tested as unknowns on 10 different days in each laboratory using the recently recommended CLSI (formerly NCCLS) disk diffusion method and the inoculum purity control method. Strains contained either macrolide-lincosamide-streptogramin B (MLSB) resistance genes encoded by erm(A) or erm(C) or a macrolide resistance efflux pump encoded by msr(A). Based upon the results of this study, strain UT 32 (now designated ATCC strain BAA-977) containing erm(A) is recommended as the positive control organism for inducible clindamycin resistance. Strain UT 25 (now designated ATCC BAA-976), which harbors the efflux pump encoded by msr(A), is recommended as the negative control organism.

Mechanisms of Linezolid Resistance among Coagulase-Negative Staphylococci Determined by Whole-Genome Sequencing

ABSTRACT Linezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ≥8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate. IMPORTANCE Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria. Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria.

Laboratory-based surveillance for vancomycin-resistant enterococci : Utility of screening stool specimens submitted for Clostridium difficile toxin assay

OBJECTIVE To study vancomycin-resistant enterococci (VRE) gastrointestinal colonization prevalence in high-risk hospitalized patients and to assess the cost and utility of this laboratory-based surveillance. SETTING Large university teaching hospital. DESIGN Quarterly prevalence culture survey of 50 stool specimens submitted for Clostridium difficile toxin A assay from October 1996 through June 1999 (n=526). Screening culture survey of all C difficile-positive stool specimens from July 1998 through June 1999 (n=140). PATIENTS Specimens for analysis were collected from patients who were admitted to the hospital and who had C difficile toxin A testing ordered. Patient samples were excluded from analysis if they were obtained from patients not hospitalized at UCLA Medical Center, if the C difficile toxin assay result was indeterminate, or if the patient was known to have previous VRE colonization or infection. RESULTS During quarterly surveillance, VRE was detected in 19.8%, C difficile toxin A in 9.5%, and both VRE and C difficile toxin A in 3.2% of stool specimens submitted for C difficile toxin assay. Patients whose stool specimens were positive for C difficile toxin A were significantly more likely than those whose specimens were negative to have VRE detected (odds ratio, 2.3; 95% confidence interval, 1.2-4.5). Based on these findings, in July 1998, we began routine screening of all C difficile-positive stool specimens for VRE. From July 1998 through June 1999, 58 (41.4%) of 140 patients with C difficile-positive specimens had VRE newly detected in the stool. The combined cost of the two laboratory-based surveillance strategies was approximately