Christine D. Steward

Pulsed-Field Gel Electrophoresis Typing of Oxacillin-Resistant Staphylococcus aureus Isolates from the United States: Establishing a National Database

ABSTRACT Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to β-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.

Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

ABSTRACT A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV andblaTEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia coli showed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.

Surveillance of Antimicrobial Use and Antimicrobial Resistance in United States Hospitals: Project ICARE Phase 2

The search for the means to understand and control the emergence and spread of antimicrobial resistance has become a public health priority. Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) has established laboratory-based surveillance for antimicrobial resistance and antimicrobial use at a subset of hospitals participating in the National Nosocomial Infection Surveillance system. These data illustrate that for most antimicrobial-resistant organisms studied, rates of resistance were highest in the intensive care unit (ICU) areas and lowest in the outpatient areas. A notable exception was ciprofloxacin- or ofloxacin-resistant Pseudomonas aeruginosa, for which resistance rates were highest in the outpatient areas. For most of the antimicrobial agents associated with this resistance, the rate of use was highest in the ICU areas, in parallel to the pattern seen for resistance. These comparative data on use and resistance among similar areas (i.e., ICU or other inpatient areas) can be used as a benchmark by participating hospitals to focus their efforts at addressing antimicrobial resistance.

Epidemiology and Clonality of Community-Acquired Methicillin-Resistant Staphylococcus aureus in Minnesota, 1996–1998

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged among patients in the general population who do not have established risk factors for MRSA. Records from 10 Minnesota health facilities were reviewed to identify cases of MRSA infection that occurred during 1996-1998 and to identify which cases were community acquired. Susceptibility testing and pulsed-field gel electrophoresis (PFGE) subtyping were performed on available isolates. A total of 354 patients (median age, 16 years) with community-acquired MRSA (CAMRSA) infection were identified. Most case patients (299 [84%]) had skin infections, and 103 (29%) were hospitalized. More than 90% of isolates were susceptible to all antimicrobial agents tested, with the exception of beta-lactams and erythromycin. Of 334 patients treated with antimicrobial agents, 282 (84%) initially were treated with agents to which their isolates were nonsusceptible. Of 174 Minnesota isolates tested, 150 (86%) belonged to 1 PFGE clonal group. CAMRSA infections were identified throughout Minnesota; although most isolates were genetically related and susceptible to multiple antimicrobials, they were generally nonsusceptible to initial empirical therapy.

gyrA Mutations Associated with Fluoroquinolone Resistance in Eight Species of Enterobacteriaceae

ABSTRACT Fluoroquinolone resistance (FQ-R) in clinical isolates ofEnterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation ingyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) ofEscherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae,Klebsiella oxytoca, Providencia stuartii, andSerratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 μg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with singlegyrA mutations in all species ofEnterobacteriaceae in this study except E. coli.

CHARACTERIZATION OF CLINICAL ISOLATES OF KLEBSIELLA PNEUMONIAE FROM 19 LABORATORIES USING THE NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS EXTENDED-SPECTRUM BETA-LACTAMASE DETECTION METHODS

ABSTRACT Extended-spectrum β-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were ≥2 μg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of ≥5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by ≥3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were ≥32 μg/ml, suggesting either the presence of an AmpC-type β-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a β-lactamase with a pI of ≥8.3 suggestive of an AmpC-type β-lactamase; 6 of the 7 isolates were shown by PCR to contain bothampC-type and blaOXA genes. The IEF profiles of the remaining 16 isolates showed a variety of β-lactamase bands, all of which had pIs of ≤7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type,blaOXA, and blaCTX-Mgenes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained β-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained β-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Testing for Induction of Clindamycin Resistance in Erythromycin-Resistant Isolates of Staphylococcus aureus

ABSTRACT Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on 128 selected nonduplicate isolates of Staphylococcus aureus. Disk diffusion testing involved placing CLI and erythromycin (ERY) disks approximately 12 mm apart (measured edge to edge) on a Mueller-Hinton agar plate that had been inoculated with an S. aureus isolate; the plate was then incubated for 16 to 18 h. Two distinct induction phenotypes (labeled D and D+) and four noninduction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) were observed in disk diffusion results. A clear, D-shaped zone of inhibition around the CLI disk was designated as the D phenotype and was observed for 21 isolates while a D-shaped zone containing inner colonies growing up to the CLI disk was designated as D+ (17 isolates). In addition, 10 isolates were CLI susceptible and ERY resistant but were not inducible and showed no blunting of the CLI zone (Neg phenotype). Isolates that were CLI and ERY resistant (constitutive macrolide-lincosamide-streptogramin B resistance) demonstrated either a double zone of inhibition with an inner ring of reduced growth up to the edge of the disks (HD phenotype; 33 isolates) or solid growth around the CLI and ERY disks (R phenotype; 16 isolates). Finally, 31 isolates were susceptible by disk testing to both CLI and ERY (S phenotype). PCR results showed that isolates with a D phenotype harbored ermA, isolates with a D+ phenotype contained either ermC (16 isolates) or ermA and ermC (one isolate), and all 10 isolates with a Neg phenotype contained msrA. All isolates with an HD or R phenotype harbored at least one erm gene. Isolates showing the D+ phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing S. aureus isolates for inducible CLI resistance.

Ability of laboratories to detect emerging antimicrobial resistance in nosocomial pathogens : a survey of Project ICARE laboratories

A proficiency testing project was conducted among 48 microbiology laboratories participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). All laboratories correctly identified the Staphylococcus aureus challenge strain as oxacillin- resistant and an Enterococcus faecium strain as vancomycin-resistant. Thirty-one (97%) of 32 laboratories correctly reported the Streptococcus pneumoniae strain as erythromycin-resistant. All laboratories testing the Pseudomonas aeruginosa strain against ciprofloxacin or ofloxacin correctly reported the organism as resistant. Of 40 laboratories, 30 (75%) correctly reported resistant MICs or zone sizes for the imipenem- and meropenem-resistant Serratia marcescens. For the extended-spectrum beta-lactamase (ESBL)-producing strain of Klebsiella pneumoniae, 18 (42%) of 43 laboratories testing ceftazidime correctly reported ceftazidime MICs in the resistant range. These results suggest that current testing generally produces accurate results, although some laboratories have difficulty detecting resistance to carbapenems and extended-spectrum cephalosporins. This highlights the need for monitoring how well susceptibility test systems in clinical laboratories detect emerging resistance.

Optimization of Computer Software Settings Improves Accuracy of Pulsed-Field Gel Electrophoresis Macrorestriction Fragment Pattern Analysis

ABSTRACT Computer-assisted analysis of pulsed-field gel electrophoresis (PFGE) libraries can facilitate comparisons of fragment patterns present on multiple gels. We evaluated the ability of the Advanced Analysis (version 4.01) and Database (version 1.12) modules of the Phoretix gel analysis software package (Nonlinear USA, Inc., Durham, N.C.) to accurately match DNA fragment patterns. Two gels containing 38 lanes of SmaI-digested Enterococcus faecalis OG1RF DNA were analyzed to assess the impact of (i) varying the lane position of the standards, (ii) using gel plugs made at different times, and (iii) normalizing the fragment patterns by using molecular weight (MW) algorithms versus retardation factor (Rf) algorithms. Two sets of PFGE libraries (one containing SmaI restriction patterns from 62 Enterococcus faecium isolates and the other containing SmaI restriction patterns of 89 Staphylococcus aureus isolates) were analyzed to assess the impact of varying the matching tolerance algorithm (designated as the vector box setting [VBS]) in the Phoretix software. Varying the lane position of standards on a gel and using gel plugs made on different days resulted in different VBSs, although it was not possible to judge whether those differences were statistically significant. Normalization of E. faecalis OG1RF fragment patterns by Rf and MW methodology yielded no statistically significant differences in variability between the same fragment on different lanes. Suboptimal VBSs decreased the specificity with which related isolates were grouped together in dendrograms. The optimal VBS for analysis of PFGE fragment patterns from E. faecalis isolates differed from that for S. aureus isolates and sometimes was not that recommended by the manufacturer. Thus, computer-assisted analysis of PFGE patterns seemed to compensate for the intra- and intergel variation evaluated in the present study, and optimizing the software for the species to be tested was a critical preliminary step before further PFGE library analysis.

Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

ABSTRACT We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.