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2.  Characterisation of a Staphylococcus aureus strain with progressive loss of susceptibility to vancomycin and daptomycin during therapy✰ 
Following an initial response to vancomycin therapy, a patient with meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia developed endocarditis, failed a second course of vancomycin and then failed daptomycin therapy. An increase in the vancomycin minimum inhibitory concentrations of four consecutive MRSA blood isolates from 2 μg/mL to 8 μg/mL was shown by Etest. Population analysis of four successive blood culture isolates recovered over the 10-week period showed that the MRSA strain became progressively less susceptible to both vancomycin and daptomycin. Retrospectively, the macro Etest method using teicoplanin indicated a decrease in vancomycin susceptibility in the second blood isolate. The patient improved after treatment with various courses of trimethoprim/sulphamethoxazole, quinupristin/dalfopristin and linezolid. Early detection of vancomycin-heteroresistant S. aureus isolates, which appeared to have clinical significance in this case, continues to be a challenge for the clinical laboratory. Development of suitable practical methods for this should be given priority. Concurrent development of resistance to vancomycin and daptomycin, whilst rare, must be considered in a patient who is unresponsive to daptomycin following vancomycin therapy.
doi:10.1016/j.ijantimicag.2008.12.010
PMCID: PMC2700752  PMID: 19233622
Staphylococci; Vancomycin; Teicoplanin; Heteroresistance
3.  Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis▿  
Journal of Clinical Microbiology  2009;47(8):2452-2457.
We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.
doi:10.1128/JCM.00476-09
PMCID: PMC2725641  PMID: 19553588
4.  Identification of Plasmid-Mediated AmpC β-Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results ▿  
Journal of Clinical Microbiology  2008;47(2):294-299.
The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum β-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC β-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC β-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.
doi:10.1128/JCM.01797-08
PMCID: PMC2643671  PMID: 19036936
5.  High Rate of Mobilization for blaCTX-Ms 
Emerging Infectious Diseases  2008;14(3):423-428.
The blaCTX-Ms have been mobilized to plasmids more frequently than other class A β-lactamases.
We constructed a phylogenetic analysis of class A β-lactamases and found that the blaCTX-Ms have been mobilized to plasmids ≈10 times more frequently than other class A β-lactamases. We also found that the blaCTX-Ms are descended from a common ancestor that was incorporated in ancient times into the chromosome of the ancestor of Kluyvera species through horizontal transfer. Considerable sequence divergence has occurred among the descendents of that ancestral gene sequence since that gene was inserted. That divergence has mainly occurred in the presence of purifying selection, which indicates a slow rate of evolution for blaCTX-Ms in the pre–antimicrobial drug era.
doi:10.3201/eid1403.070405
PMCID: PMC2570810  PMID: 18325257
CTX-M; phylogenetic analysis; plasmid; mobilization; beta-lactamase; evolution; antimicrobial resistance; test for selection; Bayesian inference; class A; research
6.  Accuracy of Six Antimicrobial Susceptibility Methods for Testing Linezolid against Staphylococci and Enterococci▿  
Journal of Clinical Microbiology  2007;45(9):2917-2922.
A challenge panel of enterococci (n = 50) and staphylococci (n = 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers’ protocols. Compared to the results of the broth microdilution method for detecting linezolid-nonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within ±1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolid-nonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.
doi:10.1128/JCM.00913-07
PMCID: PMC2045282  PMID: 17634301
7.  Multiple-Locus Variable-Number Tandem-Repeat Assay Analysis of Methicillin-Resistant Staphylococcus aureus Strains▿  
Journal of Clinical Microbiology  2007;45(7):2215-2219.
Our objective was to determine if a multiple-locus variable-number tandem-repeat assay (MLVA) for Staphylococcus aureus could predict pulsed-field gel electrophoresis (PFGE) types (i.e., USA types), thus allowing us to replace PFGE with a simpler and more rapid typing method. One hundred three well-characterized isolates representing 13 major lineages of S. aureus were tested by both PFGE and MLVA. MLVA was performed using a rapid DNA extraction technique and PCR primers for sdrCDE, clfA, clfB, sspA, and spa. PFGE was performed with genomic DNA fragments generated using SmaI, as per CDC protocols. Banding patterns were analyzed both visually and with BioNumerics software. All isolates were typeable with MLVA and PFGE. MLVA patterns were highly reproducible. PFGE separated the isolates into 13 types with 42 subtypes. Using any band difference to designate a novel MLVA type, MLVA produced 45 types, including 9 clusters containing multiple isolates. Using BioNumerics and a cutoff of >75% relatedness, MLVA produced 28 types, 11 of which contained >1 isolate. Epidemiologically related outbreak isolates of USA300-0114 from five states clustered in one MLVA pattern. USA100 isolates were present in several unrelated (<40%) MLVA types. A cutoff of >80% separated outbreak strains of USA300-0114 into three distinct MLVA types. MLVA did not differentiate community methicillin-resistant S. aureus (MRSA) lineages (USA300, USA400, USA1000, and USA1100) from health care MRSA lineages (USA100, USA200, or USA500). The ability of MLVA to differentiate among strains that are indistinguishable by PFGE may be of epidemiologic value and warrants further study.
doi:10.1128/JCM.02451-06
PMCID: PMC1932968  PMID: 17494714
8.  Comparison of Disk Diffusion, VITEK 2, and Broth Microdilution Antimicrobial Susceptibility Test Results for Unusual Species of Enterobacteriaceae▿  
Journal of Clinical Microbiology  2006;45(2):340-346.
We compared the antimicrobial susceptibility testing results generated by disk diffusion and the VITEK 2 automated system with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 61 isolates of unusual species of Enterobacteriaceae. The isolates represented 15 genera and 26 different species, including Buttiauxella, Cedecea, Kluyvera, Leminorella, and Yokenella. Antimicrobial agents included aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, and trimethoprim-sulfamethoxazole. CLSI interpretative criteria for Enterobacteriaceae were used. Of the 12 drugs tested by BMD and disk diffusion, 10 showed >95% categorical agreement (CA). CA was lower for ampicillin (80.3%) and cefazolin (77.0%). There were 3 very major errors (all with cefazolin), 1 major error (also with cefazolin), and 26 minor errors. Of the 40 isolates (representing 12 species) that could be identified with the VITEK 2 database, 36 were identified correctly to species level, 1 was identified to genus level only, and 3 were reported as unidentified. VITEK 2 generated MIC results for 42 (68.8%) of 61 isolates, but categorical interpretations (susceptible, intermediate, and resistant) were provided for only 22. For the 17 drugs tested by both BMD and VITEK 2, essential agreement ranged from 80.9 to 100% and CA ranged from 68.2% (ampicillin) to 100%; thirteen drugs exhibited 100% CA. In summary, disk diffusion provides a reliable alternative to BMD for testing of unusual Enterobacteriaceae, some of which cannot be tested, or produce incorrect results, by automated methods.
doi:10.1128/JCM.01782-06
PMCID: PMC1829079  PMID: 17135429
9.  Multicenter Evaluation of the Etest and Disk Diffusion Methods for Differentiating Daptomycin-Susceptible from Non-Daptomycin-Susceptible Staphylococcus aureus Isolates 
Journal of Clinical Microbiology  2006;44(9):3098-3104.
Daptomycin is a novel cyclic lipopeptide that is approved by the U.S. Food and Drug Administration for the treatment of complicated skin and skin structure infections associated with Staphylococcus aureus and other gram-positive pathogens and also staphylococcal bacteremia, including right-sided endocarditis. The Clinical and Laboratory Standards Institute (CLSI) established “susceptible-only” interpretive criteria for broth microdilution (BMD) and disk diffusion (DD) testing of daptomycin in 2005. However, a series of S. aureus isolates have been recovered with daptomycin MICs in the nonsusceptible range (i.e., MICs of >1 μg/ml). The objective of this study was to determine the ability of the Etest and DD methods to differentiate daptomycin-susceptible from nonsusceptible isolates of S. aureus compared to the results of the CLSI BMD reference method. There was a good correlation between Etest MIC results and the results of BMD among laboratories (r = 0.86 to 0.88), with 95.3% of the Etest MICs within a ±1 log2 dilution of the BMD MIC result. A total of 92 of 102 (90.2%) non-daptomycin-susceptible isolates of S. aureus identified by BMD in two participating laboratories were also classified as nonsusceptible by Etest. However, the very major and major error rates reported by one of the participating laboratories were 13.5 and 4.0%, respectively, primarily due to the absence of an intermediate category. The DD method, however, did not reliably differentiate daptomycin-susceptible from non-daptomycin-susceptible isolates. In 2005, daptomycin disks were voluntarily removed from the market by Cubist Pharmaceuticals. The disk diffusion breakpoints were subsequently removed from the CLSI M100 standard in 2006.
doi:10.1128/JCM.00665-06
PMCID: PMC1594672  PMID: 16954233
10.  Carbapenem Resistance in Klebsiella pneumoniae Not Detected by Automated Susceptibility Testing 
Emerging Infectious Diseases  2006;12(8):1209-1213.
Detecting β-lactamase–mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 blaKPC-positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 μg/mL to <2 μg/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems.
doi:10.3201/eid1208.060291
PMCID: PMC3291231  PMID: 16965699
carbapenem; imipenem; meropenem; susceptibility testing; Klebsiella; beta-lactamase; carbapenemase; research
11.  Class 1 Integrons in Resistant Escherichia coli and Klebsiella spp., US Hospitals 
Emerging Infectious Diseases  2006;12(6):1011-1014.
We examined Escherichia coli and Klebsiella spp. from US hospitals for class 1 integrons. Of 320 isolates, 181 (57%) were positive; association of integrons with resistance varied by drug and organism. Thus, determining integron epidemiology will improve understanding of how antibacterial resistance determinants spread in the United States.
doi:10.3201/eid1206.051596
PMCID: PMC3373057  PMID: 16707065
antimicrobial resistance; integron; Escherichia coli; Klebsiella; surveillance
12.  Testing for Induction of Clindamycin Resistance in Erythromycin-Resistant Isolates of Staphylococcus aureus 
Journal of Clinical Microbiology  2005;43(4):1716-1721.
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.
doi:10.1128/JCM.43.4.1716-1721.2005
PMCID: PMC1081368  PMID: 15814990
13.  Experimental Prediction of the Evolution of Ceftazidime Resistance in the CTX-M-2 Extended-Spectrum Beta-Lactamase 
We applied in vitro evolution to an Escherichia coli strain containing blaCTX-M-2 and obtained 10 independent mutant blaCTX-M-2 alleles that confer elevated resistance to ceftazidime (MIC ≥ 32 μg/ml) but lost the ability to confer resistance to cefepime. All alleles had a Pro-to-Ser substitution at position 167.
doi:10.1128/AAC.49.3.1242-1244.2005
PMCID: PMC549256  PMID: 15728940
14.  Utility of NCCLS Guidelines for Identifying Extended-Spectrum β-Lactamases in Non-Escherichia coli and Non-Klebsiella spp. of Enterobacteriaceae 
Journal of Clinical Microbiology  2004;42(1):294-298.
NCCLS screening and confirmation methods for detecting extended-spectrum β-lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gram-negative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was ≥2 μg/ml or the MIC of cefpodoxime was ≥8 μg/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a blaTEM, blaSHV, blaCTX-M, or blaOXA β-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested.
doi:10.1128/JCM.42.1.294-298.2004
PMCID: PMC321707  PMID: 14715768
15.  Temporal Changes in Prevalence of Antimicrobial Resistance in 23 U.S. Hospitals 
Emerging Infectious Diseases  2002;8(7):697-701.
Antimicrobial resistance is increasing in nearly all health-care–associated pathogens. We examined changes in resistance prevalence during 1996–1999 in 23 hospitals by using two statistical methods. When the traditional chi-square test of pooled mean resistance prevalence was used, most organisms appear to have increased in prevalence. However, when a more conservative test that accounts for changes within individual hospitals was used, significant increases in prevalence of resistance were consistently observed only for oxacillin-resistant Staphylococcus aureus, ciprofloxacin-resistant Pseudomonas aeruginosa, and ciprofloxacin- or ofloxacin-resistant Escherichia coli. These increases were significant only in isolates from patients outside intensive-care units (ICU). The increases seen are of concern; differences in factors present outside ICUs, such as excessive quinolone use or inadequate infection-control practices, may explain the observed trends.
doi:10.3201/eid0807.010427
PMCID: PMC2730337  PMID: 12095437
antibiotic resistance; nosocomial infections; surveillance; epidemiologic methods
16.  Monitoring Antimicrobial Use and Resistance: Comparison with a National Benchmark on Reducing Vancomycin Use and Vancomycin-Resistant Enterococci 
Emerging Infectious Diseases  2002;8(7):702-707.
To determine if local monitoring data on vancomycin use directed quality improvement and decreased vancomycin use or vancomycin-resistant enterococci (VRE), we analyzed data from 50 intensive-care units (ICUs) at 20 U.S. hospitals reporting data on antimicrobial-resistant organisms and antimicrobial agent use. We compared local data with national benchmark data (aggregated from all study hospitals). After data were adjusted for changes in prevalence of methicillin-resistant Staphylococcus aureus, changes in specific prescriber practice at ICUs were associated with significant decreases in vancomycin use (mean decrease -48 defined daily doses per 1,000 patient days, p<0.001). These ICUs also reported significant decreases in VRE prevalence compared with those not using unit-specific changes in practice (mean decrease of 7.5% compared with mean increase of 5.7%, p<0.001). In this study, practice changes focused towards specific ICUs were associated with decreases in ICU vancomycin use and VRE prevalence.
doi:10.3201/eid0807.010465
PMCID: PMC3369588  PMID: 12095438
antibiotic resistance; nosocomial infections; surveillance; benchmarking; quality
17.  Characterization of Clinical Isolates of Klebsiella pneumoniae from 19 Laboratories Using the National Committee for Clinical Laboratory Standards Extended-Spectrum β-Lactamase Detection Methods 
Journal of Clinical Microbiology  2001;39(8):2864-2872.
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 both ampC-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-M genes. 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.
doi:10.1128/JCM.39.8.2864-2872.2001
PMCID: PMC88252  PMID: 11474005
18.  Glycopeptide-Intermediate Staphylococcus aureus: Evaluation of a Novel Screening Method and Results of a Survey of Selected U.S. Hospitals 
Journal of Clinical Microbiology  1999;37(11):3590-3593.
Isolates of Staphylococcus aureus with decreased susceptibilities to glycopeptide antimicrobial agents, such as vancomycin and teicoplanin, have emerged in the United States and elsewhere. Commercially prepared brain heart infusion agar (BHIA) supplemented with 6 μg of vancomycin per ml was shown in a previous study to detect glycopeptide-intermediate S. aureus (GISA) with high sensitivity and specificity; however, this medium, when prepared in-house, occasionally showed growth of vancomycin-susceptible control organisms. This limitation could significantly impact laboratories that prepare media in-house, particularly if they wished to conduct large surveillance studies for GISA. Therefore, a pilot study to detect GISA was performed with vancomycin-containing Mueller-Hinton agar (MHA) prepared in-house in place of commercially prepared BHIA. MHA was selected for this study because this medium is widely available and well standardized. The results of the pilot study showed that supplementation of MHA with 5 μg of vancomycin per ml was both a sensitive and a specific method for screening for GISA isolates. This method was used to screen for GISA among 630 clinical isolates of methicillin-resistant S. aureus collected during 1997 from 33 U.S. hospitals. Although 14 S. aureus isolates grew on the screening agar, all were vancomycin susceptible (MICs were ≤1 μg/ml) by broth microdilution testing. Population analyses of five isolates revealed two with a subpopulation for which vancomycin MICs were 8 μg/ml. In summary, the MHA screen plate containing 5 μg of vancomycin per ml prepared in-house provides a sensitive and cost-effective method for large-scale screening for GISA for which vancomycin MICs are 8 μg/ml. However, confirmation of isolates as vancomycin resistant is critical. This study suggests that GISA was not a widespread problem in the United States in 1997.
PMCID: PMC85700  PMID: 10523558
19.  Comparison of Agar Dilution, Disk Diffusion, MicroScan, and Vitek Antimicrobial Susceptibility Testing Methods to Broth Microdilution for Detection of Fluoroquinolone-Resistant Isolates of the Family Enterobacteriaceae 
Journal of Clinical Microbiology  1999;37(3):544-547.
Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.
PMCID: PMC84460  PMID: 9986809
20.  Susceptibility of Gram-Negative Aerobic Bacilli Resistant to Carbenicillin in a General Hospital to Piperacillin and Ticarcillin 
During an 8-month period, 858 gram-negative aerobic rods resistant to carbenicillin (minimum inhibitory concentration, ≥128 μg/ml) were isolated. Susceptibility testing of 233 of these suggested that piperacillin might be active against more of these organisms than would carbenicillin or ticarcillin.
PMCID: PMC352614  PMID: 426501
21.  Susceptibility of Haemophilus influenzae Isolates from Blood and Cerebrospinal Fluid to Ampicillin, Chloramphenicol, and Trimethoprim-Sulfamethoxazole 
Susceptibility to ampicillin and chloramphenicol in vitro has been determined for Haemophilus influenzae strains isolated from blood and/or cerebrospinal fluid cultures of patients admitted to two Atlanta hospitals from 1 January 1974 to 31 March 1975. Since the appearance of ampicillin-resistant strains of this organism in early 1974, chloramphenicol has been used in these hospitals as initial therapy for severe infection due to H. influenzae. Strains from five of 94 patients were resistant to ampicillin (minimum inhibitory concentration [MIC] ≥ 12.5 μg/ml), but all strains were susceptible to chloramphenicol (MIC < 2 μg/ml). The first 35 strains studied, including three resistant to ampicillin, were also tested for in vitro susceptibility to trimethoprim-sulfamethoxazole; all were highly susceptible (MIC ≤ 0.0312 μg of trimethoprim and 0.625 μg of sulfamethoxazole per ml).
PMCID: PMC429488  PMID: 1083198
22.  Rapid Semiquantitative Testing of Antibiotic Susceptibility: Use of a Multicell Disk Elution System 
Semiquantitative data regarding antibiotic susceptibility of bacteria were obtained by measuring the growth of bacteria in several different compartments (“cells”) of a plastic module. Each compartment contained an inoculum of the organism and various concentrations of antibiotics in broth, achieved by elution of antibiotic from paper disks placed into the individual cells. Growth of the organisms was measured using an automated monitor to detect the scattering of light. Susceptibility of 300 strains of gram-negative bacilli to multiple concentrations of nine antibiotics was determined by this disk elution system, and the results (expressed in terms of four clinical susceptibility groupings) were compared with those obtained by a quantitative agar dilution method. Results obtained by the two methods agreed completely in 78% of the 2,700 determinations. In evaluating whether individual strains would be susceptible to systemic therapy or not, results obtained by the two systems agreed in all except 149 of the tests. Results of testing by the disk elution method were available on the same day that testing was begun. A system of this type may prove useful, for it provides information of a semiquantitative nature and decreases the time between isolation of a bacterial pathogen and availability of susceptibility data to the clinician.
PMCID: PMC429180  PMID: 1147587
24.  SOME OBSERVATIONS ON RICKETS 
British Medical Journal  1933;2(3795):599-612.4.
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PMCID: PMC2369264  PMID: 20777799

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