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Daptomycin is one of the few treatment options for infections caused by enterococci that are resistant to ampicillin and vancomycin, such as vancomycin-resistant Enterococcus faecium. The emergence and clinical significance of daptomycin-resistant enterococci and evolving microbiologic, pharmacokinetic-pharmacodynamic, and clinical data indicated that the pre-2019 Clinical and Laboratory Standards Institute (CLSI) susceptible-only breakpoint of ≤4 μg/mL for daptomycin and enterococci was no longer appropriate. After analyzing data that are outlined in this article, the CLSI Subcommittee on Antimicrobial Susceptibility Testing established new breakpoints for daptomycin and enterococci. For E. faecium, a susceptible dose-dependent (SDD) breakpoint of ≤4 μg/mL was established based on an increased dosage of 8–12 mg/kg/day (≥8 μg/mL-resistant). CLSI suggests infectious diseases consultation to guide daptomycin use for the SDD category. For Enterococcus faecalis and other enterococcal species, revised breakpoints of ≤2 μg/mL-susceptible, 4 μg/mL-intermediate, and ≥8 μg/mL-resistant were established based on a standard dosage of 6 mg/kg/day.

Purpose To assess the potency and sterility of ophthalmic antibiotic drops commonly used in the treatment of bacterial keratitis. Methods This was a basic investigation. Three drugs were tested: fortified vancomycin 25 mg/mL, fortified tobramycin 14 mg/mL, and moxifloxacin 5 mg/mL. A bottle of each was stored separately at 4, 24, and 35°C, with the potency determined by microbiological assay at 0, 7, and 14 days. Differences in potency were assessed by 2-way analysis of variance followed by a 1-way repeated-measures analysis of variance with Bonferroni post hoc testing as warranted. Sterility of drugs when handled by patients for varying periods was confirmed by culturing samples on MacConkey and sheep blood agars. Results The concentration of fortified tobramycin and moxifloxacin remained constant over 14 days at the 3 tested temperatures. The concentration of fortified vancomycin remained constant at 4°C, but it declined by 38% ± 1% (P = 0.001) at 24°C on day 14 and by 48% ± 1% (P = 0.001) and 78% ± 3% (P = 0.0009) at 35°C on days 7 and 14, respectively. A total of 49 drops (mean, 7.3 days; range, 1-18 days) were tested for sterility, and all were negative for microbial contamination. Conclusions All 3 drugs remained potent at 4°C for up to 14 days. Fortified tobramycin and moxifloxacin also maintained potency for 14 days at 24 and 35°C. In contrast, fortified vancomycin lost its potency by day 14 at 24°C and by day 7 at 35°C. All in-use antibiotic drops tested were sterile. The results indicate that patients should be cautioned to store vancomycin under refrigerator or at least under cool conditions.

Clinical microbiology is in the midst of an era of emphasis on rapid test performance and reporting, often through use of automated instrumentation. Results which are generated very rapidly using an automated instrument, but which are not available in the patient's medical record until the following day cannot improve clinical decision making. A number of clinical microbiology laboratories employ computers ranging in size from small personal or microcomputers which are packaged as instrument-based "data management" systems to large laboratory-wide main frame systems which may include on-line interfacing of certain automated microbiology instruments. Results generated by automated instruments offer the greatest potential for rapid result transmission by directly interfacing the microcomputer which controls the instrument's function with the microbiology host computer. Computer generation of reports for use by those outside the laboratory is the final and often most visible step in use of computers in clinical microbiology.

Setting Five health care systems in Texas. Objective To describe the epidemiology of inadequate isolation for pulmonary tuberculosis leading to tuberculosis (TB) exposures from confirmed TB patients and the patient factors that led to the exposures. Design A retrospective cohort and case-control study of adult patients with TB resulting in exposures (cases) vs those TB patients who did not result in exposures (controls) during January 2005 to December 2012. Results There were 335 patients with pulmonary TB disease, 199 cases and 136 controls. There was no difference between groups in age (46 ± 14.6 vs 45 ± 17 years; P > .05), race, or substance abuse. Cases were more likely to be transplant recipients (adjusted odds ratio [AOR], 18.90; 95% CI, 1.9–187.76), have typical TB chest radiograph (AOR, 2.23; 95% CI, 1.1–4.51), and have positive acid-fast bacilli stains (AOR, 2.36; 95% CI, 1.31–4.27). Cases were less likely to have extrapulmonary disease (AOR, 0.47; 95% CI, 0.24–0.95). Conclusions TB exposure resulting from inadequate isolation is frequent in health care settings. Extrapulmonary involvement resulted in earlier airborne isolation. Being a transplant recipient, having chest radiograph findings typical for TB, and sputum positivity acid-fast bacilli upon staining were associated with increased risk of inadequate isolation.

Background. A clinical trial of mass azithromycin distributions for trachoma created a convenient experiment to test the hypothesis that antibiotic use selects for clonal expansion of preexisting resistant bacterial strains. Methods. Twelve communities in Ethiopia received mass azithromycin distributions every 3 months for 1 year. A random sample of 10 children aged 0–9 years from each community was monitored by means of nasopharyngeal swab sampling before mass azithromycin distribution and after 4 mass treatments. Swab specimens were tested for Streptococcus pneumoniae, and isolates underwent multilocus sequence typing. Results. Of 82 pneumococcal isolates identified before treatment, 4 (5%) exhibited azithromycin resistance, representing 3 different sequence types (STs): 177, 6449, and 6494. The proportion of isolates that were classified as one of these 3 STs and were resistant to azithromycin increased after 4 mass azithromycin treatments (14 of 96 isolates [15%]; P = .04). Using a classification index, we found evidence for a relationship between ST and macrolide resistance after mass treatments (P < .0001). The diversity of STs—as calculated by the unbiased Simpson index—decreased significantly after mass azithromycin treatment (P = .045). Conclusions. Resistant clones present before mass azithromycin treatments increased in frequency after treatment, consistent with the theory that antibiotic selection pressure results in clonal expansion of existing resistant strains.

Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo . A. baumannii strain CI 79 exhibited significantly ( P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD 50 ) of 10 5 CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.

Children with sickle cell disease (SCD) are at increased risk of illness and death from invasive pneumococcal disease (IPD). The introduction in 2000 of the 7-valent pneumococcal conjugate vaccine and penicillin prophylaxis for children with SCD has greatly reduced the incidence of IPD in this population. However, a recent report suggested an increase in cases of IPD in children with SCD.Using data from Active Bacterial Core surveillance, we analyzed trends in hospitalizations, mortality and serotype among children with SCD compared with other children. We used neonatal screening data to estimate SCD population denominators for each Active Bacterial Core surveillance site.From 1998 to 2009, 3069 cases of IPD occurred among African-American children less than 18 years of age in the Active Bacterial Core surveillance catchment area. Of these, 127 (4.1%) had SCD identified by medical chart review and 185 (6.0%) had 1 or more IPD risk factors, excluding SCD. Rates of IPD among children with SCD declined by 53% (1118 vs. 530 per 100,000) whereas the overall rates among African-American children declined by 74% (54 to 14 per 100,000). For all time periods, children with SCD and IPD were more likely to be hospitalized (84%-92% vs. 31%-56%) and more likely to die (6%-17% vs. 1%-2%) than children with no risk factors.Although the rate of IPD in children with SCD has dropped dramatically since 7-valent pneumococcal conjugate vaccine introduction, the rate of IPD in children with SCD remains higher than that of the general population of African-American children, pointing to the need for more effective prevention efforts to prevent IPD in children with SCD.

In 2009, Streptococcus pneumoniae, or pneumococcus, caused approximately 44 000 cases of invasive pneumococcal disease (IPD) and about 5000 deaths in the United States [1]. In addition, pneumococcus is responsible for a considerable burden of respiratory disease globally [2]. Antibiotic nonsusceptibility is a major concern; understanding the dynamics by which pneumococcus develops nonsusceptibility will help maintain the efficacy of these drugs. There are currently over 90 known serotypes of S. pneumoniae, of which only a small proportion account for most invasive disease and most nonsusceptible infections in the United States. Before introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the United States in 2000, the proportion of penicillin-nonsusceptible and macrolide-nonsusceptible isolates from invasive disease was increasing [3–5]. Previous studies have correlated differential use of antimicrobials with different rates of nonsusceptibility across geographic areas, as well as changing patterns of usage and nonsusceptibility over time [6–11]. When PCV7 was introduced, the 7 serotypes included in the vaccine accounted for 78% of penicillin-nonsusceptible strains [3]. In the decade since, rates of disease caused by these 7 serotypes declined by 99%, and serotypes not included in PCV7 have increased significantly, a phenomenon known as serotype replacement. Some of the replacing serotypes (eg, 19A, 15A, 23A, 35B, 6C) have shown high and/or increasing levels of antimicrobial nonsusceptibility, raising the question of whether antimicrobial use may in part be responsible for the increased prevalence [12–14]. In the pre-PCV7 era, geographic differences in proportions of nonsusceptibility to penicillin and macrolides resulted from geographic variation in selective pressure for nonsusceptibility [10, 15]. In pneumococci, selective pressure may affect nonsusceptibility in 2 ways: by increasing the prevalence of nonsusceptibility within each serotype, and/or by increasing the prevalence of serotypes in which the prevalence of nonsusceptibility is high. The frequencies of pneumococcal serotypes within a population reflect multiple selective pressures imposed by natural and vaccine-induced host immunity [16], and it is in principle possible that selection by antimicrobial use may also affect this pattern. In the pre-PCV7 era, no evidence of such an effect was found; the impact of differences in antimicrobial selective pressure was seen solely in varying prevalence of nonsusceptibility within each serotype, but not in variations in the prevalence of the more nonsusceptible serotypes [15]. Understanding the interplay between antimicrobial use pressures and changing serotype distributions has important policy implications for both antimicrobial stewardship and vaccination programs. In this study, we assessed geographical, serotype, and temporal variation in the frequency of drug nonsusceptibility, with a particular focus on the question of whether, contrary to pre-PCV7 observations, there was evidence that geographic variation in the intensity of selective pressure for nonsusceptibility was reflected in greater prevalence of nonsusceptible serotypes.

Streptococcus anginosus is a member of the normal oral flora that can become a pathogen causing pyogenic infections in humans. The genome of daptomycin-resistant strain J4206, originally isolated from a patient suffering from breakthrough bacteremia and septic shock at the University of Texas Health Science Center at San Antonio, was determined. The circular genome is 2,001,352 bp long with a GC content of 38.62% and contains multiple mobile genetic elements, including the phage-like chromosomal island SanCI that mediates a mutator phenotype, transposons, and integrative conjugative elements. Daptomycin resistance involves multiple alterations in the cell membrane and cell wall, and unique features were identified in J4206 that may contribute to resistance. A cluster of capsular polysaccharide (CPS) genes for choline metabolism and transport are present that may help neutralize cell surface charges, destabilizing daptomycin binding. Further, unique J4206 genes encoding sortases and LPXTG-target proteins that are involved in cell wall modification were present. The J4206 genome is phylogenetically closely related to the recently reported vancomycin-resistant SA1 strain; however, these genomes differ with SNPs in cardiolipin synthetase, histidine kinase yycG, teichoic acid modification genes, and other genes involved in cell surface modification. Transmission electron microscopy showed that the cell walls of both strains J4206 and SA1 were significantly thicker and more electron dense than daptomycin- and vancomycin-sensitive strain J4211. This comparative genomic study has identified unique genes as well as allelic variants in the J4206 genome that are involved in cell surface modification and thus might contribute to the acquisition of daptomycin resistance.

Insulin-dependent type 1 diabetes mellitus (DM) and oral diseases are closely interrelated. Poor metabolic control in diabetics is associated with a high risk of gingivitis, periodontitis and tooth loss. Salivary flow declines in diabetics and patients suffer from xerostomia. Reduced saliva predisposes to enamel hypomineralization and caries formation; however, the mechanisms that initiate and lead to progression of tooth decay and periodontitis in type 1 DM have not been explored. To address this issue, we analyzed tooth morphology in Akita −/− mice that harbor a point mutation in the Ins2 insulin gene, which leads to progressive hyperglycemia. Mandibles from Akita −/− and wild-type littermates were analyzed by microCT, scanning EM and histology; teeth were examined for amelogenin (Amel) and ameloblastin (Ambn) expression. Mice were injected with pilocarpine to assess saliva production. As hyperglycemia may alter pulp repair, the effect of high glucose levels on the proliferation/differentiation of cultured MD10-F2 pulp cells was also analyzed. Results showed that Akita −/− mice at 6 weeks of age showed chalky white incisors that correlated with marked hyperglycemia and impaired saliva production. MicroCT of Akita −/− teeth revealed excessive enamel wearing and hypomineralization; immunostaining for Amel and Ambn was decreased. A striking feature was invasion of dentinal tubules with Streptococcus mitis and microabcesses that originated in the coronal pulp and progressed to pulp necrosis and periapical periodontitis. High levels of glucose also inhibited MD10-F2 cell proliferation and differentiation. Our findings provide the first evidence that hyperglycemia in combination with reduced saliva in a model of type1 DM leads to decreased enamel mineralization/matrix proteins and predisposes to excessive wearing and decay. Importantly, hyperglycemia adversely affects enamel matrix proteins and pulp repair. Early detection and treatment of hyperglycemia and hyposalivation may provide a useful strategy for preventing the dental complications of diabetes and promoting oral health in this population.

We investigated the clinical and microbiologic features of 300 cases of cephalosporin-resistant Escherichia coli producing extended-spectrum β-lactamase (ESBL) or plasmid-mediated AmpC β-lactamase (pAmpC) at three medical centers in the United States. Solid-organ malignancy, connective tissue disease, and a recent history of surgery were more common among pAmpC-producing cases ( n = 49), whereas urinary catheter at enrollment, diabetes, and hospitalization in the past year were more common among ESBL-producing cases ( n = 233). The factors independently associated with clinical outcome were the following: the presence of cardiovascular disease (odds ratio [OR], 2.88; 95% confidence interval [CI], 1.29 to 6.43), intra-abdominal infection (OR, 6.35; 95% CI, 1.51 to 26.7), other or multiples sources of infection (OR, 8.12; 95% CI, 2.3 to 28.6), age of 65 years or greater (OR, 0.43; 95% CI, 0.2 to 0.95), favorable baseline health status (OR, 0.39; 95% CI, 0.16 to 0.95), and appropriate empirical antimicrobial therapy given in the first 72 h (OR, 0.42; 95% CI, 0.20 to 0.88). β-Lactamase genes responsible for cephalosporin resistance were identified in 291 cases. CTX-M-type ESBLs accounted for 72.0%. Of those, 88.0% were CTX-M-15. The next most common type was CMY-type pAmpC (16.7%), followed by SHV- and TEM-type ESBLs (6.3 and 1.3%, respectively). Seven cases (2.3%) had KPC-type β-lactamase. Ertapenem, imipenem, meropenem, doripenem, piperacillin-tazobactam, amikacin, nitrofurantoin, and tigecycline were highly active, with greater than 90% of the isolates being susceptible. Cefepime was less active, with only 74.2% being susceptible due to the predominance of CTX-M-15. These findings have implications in the selection of appropriate empirical therapy when infection due to cephalosporin-resistant E. coli is suspected.

Extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacteriaceae are often resistant to multiple drug classes, making therapy of urinary infections with oral antibiotics difficult. Previously it was shown that amoxicillin-clavulanate can provide clavulanate inhibition of ESBLs and protect an oral cephalosporin present in combination when tested by broth microdilution. This study has shown that disk approximation testing could detect favorable cephalosporin-clavulanate interactions among a group of 101 previously characterized members of the Enterobacteriaceae with CTX-M, SHV, or TEM ESBLs.

Background. Streptococcus pneumoniae (pneumococcus) caused approximately 44000 US invasive pneumo-coccal disease (IPD) cases in 2008. Antibiotic nonsusceptibility complicates IPD treatment. Using penicillinsusceptibility breakpoints adopted in 2008, we evaluated antibiotic-nonsusceptible IPD trends in light of theintroductions of a 7-valent pneumococcal conjugate vaccine (PCV7) in 2000 and a 13-valent pneumococcalconjugate vaccine (PCV13) in 2010.Methods. IPD cases were defined by isolation of pneumococcus from a normally sterile site in individualsresiding in Active Bacterial Core surveillance (ABCs) areas during 1998–2008. Pneumococci were serotyped andtested for antibiotic susceptibility using broth microdilution.Results. During 1998–2008, ABCs identified 43198 IPD cases. Penicillin-nonsusceptible strains caused 6%–14%of IPD cases, depending on age. Between 1998–1999 and 2008, penicillin-nonsusceptible IPD rates declined 64% forchildrenaged,5years(12.1–4.4casesper100000),and45%foradultsaged$65(4.8–2.6casesper100000).RatesofIPD nonsusceptible to multiple antibiotics mirrored these trends. During 2007–2008, serotypes in PCV13 but notPCV7 caused 78%–97% of penicillin-nonsusceptible IPD, depending on age.Conclusions. Antibiotic-nonsusceptible IPD rates remain below pre-PCV7 rates for children ,5 and adults$65 years old. PCV13 vaccines hold promise for further nonsusceptibility reductions.Streptococcus pneumoniae (pneumococcus) caused ap-proximately 63000 invasive pneumococcal disease(IPD)casesannuallyinthelate1990sintheUnitedStates,leading to about 6100 deaths [1]. During the first7 years after the introduction in the United States of a7-valent pneumococcal conjugate vaccine (PCV7) forchildren, an estimated 211000 fewer cases of IPD oc-curredamongallagesthanwouldhaveoccurredwithoutthe vaccine [2]; however, approximately 44000 IPDcasescontinuetooccurannually[3].Antibioticresistanceand intermediate susceptibility, together termed non-susceptibility,complicatemanagementofpneumococcaldisease [4–6]. Despite increasing during the 1990s [7],the incidence of antibiotic-nonsusceptible IPD in theUnited States fell following the introduction of PCV7[8, 9]. The 7 serotypes covered by PCV7 accounted for78% of nonsusceptible serotypes in 1998 [7], and theincidence rate of these serotypes decreased 78% amongchildren aged ,2 years by 2001 [9]. However, by 2003,the incidence of antibiotic-nonsusceptible IPD in chil-dren aged ,5 years was increasing again [8], coincidingwith the emergence of serotypes not included in PCV7

This chapter on special phenotypic methods for detecting antibacterial resistance describes details of tests for detection of high-level aminoglycoside resistance and acquired vancomycin resistance in enterococci; tests for detection of inducible clindamycin resistance in streptococci; tests for detection of penicillin, oxacillin, vancomycin, induc-ible clindamycin, and high-level mupirocin resistance in staphylococci; tests for detection of extended-spectrum-β-lactamase (ESBL) and carbapenemase production in Enterobacteriaceae; and tests for detection of β-lactamases in multiple organisms. Other special phenotypic tests described in the chapter are those for determining the bactericidal activity or combined activities of antimicrobial agents; they are different from the tests that screen for a specific resistance mechanism. As some of the phenotypic tests described here for Staphylococcus aureus are not recommended for coagulase-negative staphylococci (CoNS), for detection of oxacillin resistance, the two groups are discussed separately as S. aureus and Staphylococcus lugdunensis and CoNS except S. lugdunensis. In the clinical laboratory, β-lactamase tests can be used for two purposes. The first is to detect an underlying mechanism of resistance that may not be detected using routine susceptibility testing methods, and the second is to detect a mechanism of resistance that is an infection control concern and epidemiologically important.

Most fastidious bacteria do not grow satisfactorily in standard in vitro susceptibility test systems that use unsupple-mented media. Clinical and Laboratory Standards Institute (CLSI) has also published an approved guideline for testing infrequently isolated or fastidious bacteria including Abiotrophia spp., Granulicatella spp., Aeromonas spp., Plesiomonas spp., Bacillus spp. (not B. anthracis), Campylobacter jejuni/coli, Corynebacterium spp., Erysipelothrix rhusiopathiae, the HACEK group, Lactobacillus spp., Leuconostoc spp., Listeria monocytogenes, Moraxella catarrhalis, Pasteurella spp., Pediococcus spp., and Vibrio spp. In addition to conventional MIC test methods (e.g., agar dilution or broth dilution methods), the Etest MIC determination method has been used to test many types of fastidious bacteria. The limitations of this method include its cost and lack of clearance by the U.S. Food and Drug Administration (FDA) for testing many less commonly encountered fastidious bacteria. This chapter summarizes the standard methods recommended by CLSI for antimicrobial susceptibility testing of Streptococcus spp. (including S. pneumoniae), H. influenzae, N. gonorrhoeae, and N. meningitidis. Methods for testing the infrequently isolated or fastidious bacteria included in the CLSI M45 guideline are summarized to include testing potential agents of bioterrorism. The incidence of resistance, test methods, and indications for testing and the reporting of results are provided.

G.R. Thompson III, B.L. Wickes, M.L. Herrera, T.C. Haman, J.S. Lewis II, J.H. Jorgensen. Disseminated Burkholderia gladioli infection in a lung transplant recipient with underlying hypocomplementemic urticarial vasculitis. Transpl Infect Dis 2011: 13: 641–645. All rights reserved Abstract: Burkholderia gladioli is difficult to definitively identify within the laboratory using phenotypic testing alone. We describe a case of recurrent B. gladioli infection in a lung transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome, discuss the difficulties encountered with laboratory identification, provide a review of the methodology required for definitive identification, and discuss potential pathophysiologic mechanisms in this patient responsible for the difficulty in treatment.

CTX-M extended-spectrum beta-lactamases (ESBLs) have emerged as the most common type of ESBL globally, their incidence easily surpassing those of SHV and TEM ESBLs in most locales. This study compared the performance of two MicroScan dried panels with CLSI reference broth microdilution and disk diffusion methods on a collection of genetically characterized ESBL-producing isolates. These included 64 Enterobacteriaceae isolates that produced CTX-M8, -14, -15, or -16 according to PCR and sequencing of the bla gene, 17 isolates that produced a SHV or TEM ESBL, and 19 that produced both CTX-M and SHV ESBLs. Each isolate was tested by a frozen reference microdilution panel, the MicroScan ESβL plus confirmation panel, and a routine dried panel containing streamlined ESBL confirmation dilutions (MicroScan Neg MIC panel type 32) that included cefotaxime and ceftazidime tested alone or with a fixed concentration of 4 μg/ml of clavulanate. Each isolate was also tested by the standard CLSI double-disk confirmation tests. The disk diffusion method detected all ESBL-producing isolates, the frozen reference panel detected 90% of isolates (10 out of 100 could not be analyzed because of off-scale MICs that exceeded the clavulanate combination concentrations in the panel), the ESβL plus panel detected 98% (1 missed and 1 off scale), and the streamlined ESBL panel detected 95% (5 off scale). Very high MICs for a few strains that produced SHV or both CTX-M and SHV ESBLs precluded noting the required three twofold-dilution differences with clavulanate needed to confirm an ESBL primarily in the reference panel and the Neg type 32 panel.

An important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. The most widely used testing methods include broth microdilution or rapid automated instrument methods that use commercially marketed materials and devices. Manual methods that provide flexibility and possible cost savings include the disk diffusion and gradient diffusion methods. Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results (eg, minimum inhibitory concentration), and all provide qualitative assessments using the categories susceptible, intermediate, or resistant. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms. However, newer or emerging mechanisms of resistance require constant vigilance regarding the ability of each test method to accurately detect resistance.

BACKGROUND: Serotype 19A invasive pneumococcal disease (IPD) increased annually in the United States after the introduction of the 7-valent conjugate vaccine (PCV7). To understand this increase, we characterized serotype 19A isolates recovered during 2005. METHODS: IPD cases during 1998-2005 were identified through population-based surveillance. We performed susceptibility testing and multilocus sequence typing on 528 (95%) of 554 serotype 19A isolates reported in 2005. RESULTS: The incidence of IPD due to serotype 19A increased from 0.8 to 2.5 cases per 100,000 population between 1998 and 2005 (P < .05), whereas the overall incidence of IPD decreased from 24.4 to 13.8 cases per 100,000 population (P < .05). Simultaneously, the incidence of IPD due to penicillin-resistant 19A isolates increased from 6.7% to 35% (P < .0001). Of 151 penicillin-resistant 19A isolates, 111 (73.5%) belonged to the rapidly emerging clonal complex 320, which is related to multidrug-resistant Taiwan(19F)-14. The remaining penicillin-resistant strains were highly related to other clones of PCV7 serotypes or to isolates within major 19A clonal complex 199 (CC199). In 1999, only CC199 and 3 minor clones were apparent among serotype 19A isolates. During 2005, 11 multiple-isolate clonal sets were detected, including capsular switch variants of a serotype 4 clone. CONCLUSIONS: PCV7 ineffectiveness against serotype 19A, antibiotic resistance, clonal expansion and emergence, and capsular switching have contributed to the genetic diversity of 19A and to its emergence as the predominant invasive pneumococcal serotype in the United States.

We assessed MICs and minimal bactericidal concentrations of vancomycin, daptomycin, and nine other antimicrobials against methicillin-resistant Staphylococcus aureus isolates obtained from 1999 through 2006. No vancomycin, daptomycin, or linezolid resistance was observed. Clindamycin, gentamicin, and ciprofloxacin resistance decreased significantly. No tolerance to vancomycin or daptomycin was observed, nor was MIC creep seen.

Ceftobiprole MICs at which 50% and 90% of isolates were inhibited (MIC50 and MIC90), determined by the Clinical and Laboratory Standards Institute broth microdilution method, were both 1 microg/mL (range, 0.5-1 microg/mL) against 143 community-associated methicillin-resistant Staphylococcus aureus isolates and 0.5 microg/mL (range, 0.25-0.5 microg/mL) with 29 methicillin-susceptible isolates recovered from military trainees during 2 prospective investigations.

This study used pharmacokinetic-pharmacodynamic (PK-PD) modelling and MICs of 15 antimicrobial agents, derived from testing a large international culture collection, to assist in the development of interpretative criteria, i.e., breakpoints, for Neisseria meningitidis. PK parameters, protein binding, percentage penetration into cerebrospinal fluid (CSF), and the variability of these values, were extracted from the published literature for the 15 agents. PK-PD parameters have not been developed specifically for N. meningitidis in animal or human studies. Thus, it was necessary to invoke PK-PD targets from other organisms that cause infections at similar sites. The PK-PD targets utilised were: time above the MIC for at least 50% of the dosing interval for all beta-lactams, chloramphenicol, sulphafurazole and trimethoprim-sulphamethoxazole; an AUC/MIC ratio of >or=25 for the tetracyclines and macrolides; and an AUC/MIC ratio of >or=125 for the fluoroquinolones. A 10 000-subject Monte Carlo simulation was designed with the usual dosing regimens of each antimicrobial agent at MIC values of 0.03-64 mg/L in both serum and CSF. The PK-PD breakpoint was defined as the MIC at which the calculated target attainment was >or=95%. Using these assumptions, the proposed PK-PD breakpoints were: azithromycin, 0.125 mg/L; doxycycline, 0.25 mg/L; cefotaxime, ciprofloxacin and levofloxacin, 0.5 mg/L; penicillin G, meropenem, rifampicin, tetracycline and minocycline, 1 mg/L; chloramphenicol and sulphafurazole, 2 mg/L; and ampicillin, ceftriaxone and trimethoprim-sulphamethoxazole, 4 mg/L. Proposed PK-PD breakpoints applicable to CSF were: penicillin and cefotaxime, 0.06 mg/L; rifampicin, 0.125 mg/L; ceftriaxone, meropenem and trimethoprim-sulphamethoxazole, 0.25 mg/L; ampicillin, 0.5 mg/L; and chloramphenicol, 1 mg/L.

Summary Background In 2010, 13-valent pneumococcal conjugate vaccine (PCV13) was licensed and recommended in the USA for prevention of invasive pneumococcal disease in children. Licensure was based on immunogenicity data comparing PCV13 with the earlier seven-valent formulation. Because clinical endpoints were not assessed for the new antigens, we did a postlicensure matched case-control study to assess vaccine effectiveness. Methods Cases in children aged 2–59 months were identified through active surveillance in 13 sites. Controls were identified via birth registries and matched to cases by age and postal (zip) code. The primary objective was the vaccine effectiveness of at least one dose against the 13 serotypes included in PCV13. Secondary objectives included vaccine effectiveness against all-cause invasive pneumococcal disease, against antibiotic non-susceptible invasive pneumococcal disease, and among children with and without underlying conditions. Vaccine effectiveness was calculated as (1 – matched odds ratio) × 100%. Findings We enrolled 722 children with invasive pneumococcal disease and 2991 controls; PCV13 serotype cases (217 [30%]) included most commonly serotypes 19A (128 [18%]), 7F (32 [4%]), and 3 (43 [6%]). Vaccine effectiveness against PCV13 serotypes was 86·0% (95% CI 75·5 to 92·3), driven by serotypes 19A and 7F, for which vaccine effectiveness was 85·6% (95% CI 70·6 to 93·5) and 96·5% (82·7 to 100), respectively. We also identified statistically significant effectiveness against serotype 3 (79·5%, 95% CI 30·3 to 94·8) and against antibiotic non-susceptible invasive pneumococcal disease (65·6%, 44·9 to 78·7). Vaccine effectiveness against all-cause invasive pneumococcal disease was 60·2% (95% CI 46·8 to 70·3). Vaccine effectiveness was similar among children with (81·4%, 95% CI 45·4 to 93·6) and without (85·8%, 74·9 to 91·9) underlying conditions. Interpretation PCV13 appears highly effective against invasive pneumococcal disease among children in the USA in the context of routine and catch-up schedules, although some new vaccine antigens could not be assessed. PCV13 immunisation provides a robust strategy for combating pneumococcal antimicrobial resistance. Funding Centers for Disease Control and Prevention.

Streptococcus anginosus is an opportunistic human pathogen that causes abscesses of the brain, liver, and other organs. Here, we announce the complete genome sequence of a clinically isolated strain of S. anginosus J4211. The genome sequence contains two prophages and multiple mobile genetic elements.

This chapter by the co-editors in chief summarizes the major changes in the field of clinical microbiology since the last edition and lists highlights of the 11th edition of the Manual. It recognizes the important contributions of the volume editors, section editors, and authors who have contributed to this edition.

This chapter describes reference dilution (MIC) and disk diffusion antimicrobial susceptibility test methods for nonfastidious, aerobic bacteria. It describes CLSI (United States) and EUcst (European) reference methods and standardized disk diffusion methods. The procedure for performing each test type is described as well as quality control procedures. The advantages and disadvantages of each method are discussed. Finally, the most current interpretive breakpoint criteria for MIC and disk tests from the CLSI are listed.

and their disease processes based on evaluation of disease signs and symptoms. As with many infectious disease syndromes, a zoonotic infection often debuts as a nonspecific febrile illness, and a discussion of a common approach to the patient who presents with a nonspecific febrile illness could potentially help clinicians identify specific disease processes in a costefficient and time-efficient manner. The current book format requires that the reader already have some basic knowledge of the specific disease-causing agent(s) and the differential diagnoses for the disease syndrome. There are some minor inaccuracies in a number of the subchapters throughout the book. For example, we know now that 1 of the 3 hunters in Northern Wisconsin who allegedly died of CreutzfeldtJakob disease actually died of Pick disease, which has not been related to prion disease. Chloramphenicol is no longer recommended for treatment of human ehrlichial or anaplasma infections, given the lack of demonstrated in vitro effect and clinical failure in vivo resulting in fatal outcomes for several children. The mortality rate for patients with human anaplasmosis was initially estimated to be as high as 15%. More recent information indicates that the rate is 1% or lower. The impact of these and other inaccuracies on the reading experience as a whole, however, is minor. Abbreviations are frequently used throughout the text, and a short glossary of commonly used laboratory terms can be found in the introductory pages. An expanded glossary that also includes the specific disease names and their abbreviations (such as Japanese encephalitis [JE], St. Louis encephalitis [SLE], human monocytic ehrlichiosis [HME], and others) would have enhanced and expedited the reading experience. Each of the subchapters concludes

Summary Background In 2000, seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in the USA and resulted in dramatic reductions in invasive pneumococcal disease (IPD) and moderate increases in non-PCV7 type IPD. In 2010, PCV13 replaced PCV7 in the US immunisation schedule. We aimed to assess the effect of use of PCV13 in children on IPD in children and adults in the USA. Methods We used laboratory-based and population-based data on incidence of IPD from the Active Bacterial Core surveillance (part of the Centers for Disease Control and Prevention's Emerging Infections Program) in a time-series model to compare rates of IPD before and after the introduction of PCV13. Cases of IPD between July 1, 2004, and June 30, 2013, were classified as being caused by the PCV13 serotypes against which PCV7 has no effect (PCV13 minus PCV7). In a time-series model, we used an expected outcomes approach to compare the reported incidence of IPD to that which would have been expected if PCV13 had not replaced PCV7. Findings Compared with incidence expected among children younger than 5 years if PCV7 alone had been continued, incidence of IPD overall declined by 64% (95% interval estimate [95% IE] 59–68) and IPD caused by PCV13 minus PCV7 serotypes declined by 93% (91–94), by July, 2012, to June, 2013. Among adults, incidence of IPD overall also declined by 12–32% and IPD caused by PCV13 minus PCV7 type IPD declined by 58–72%, depending on age. We estimated that over 30 000 cases of IPD and 3000 deaths were averted in the first 3 years after the introduction of PCV13. Interpretation PCV13 reduced IPD across all age groups when used routinely in children in the USA. These findings provide reassurance that, similar to PCV7, PCVs with additional serotypes can also prevent transmission to unvaccinated populations. Funding Centers for Disease Control and Prevention.

The International Organization for Standardization (ISO) has recently provided an opportunity to define a global standard reference method for antimicrobial susceptibility testing and to delineate acceptable performance criteria for antimicrobial susceptibility testing devices. A working group organized under the auspices of ISO Technical Committee 212 has worked closely with a working group of the European Committee on Normalization to develop the two global standards. The global reference method is the broth microdilution method that is presently described by both the Clinical and Laboratory Standards Institute and the European Union Committee on Antimicrobial Susceptibility Testing. Experts from 13 countries that formed the membership of the working groups have endeavored over the past 3 years to agree upon the international reference method and to define for the first time an outline for objective assessment of commercial devices that can be used for routine susceptibility testing in clinical microbiology laboratories.

Summary Background When seven-valent pneumococcal conjugate vaccine was introduced in the USA, many children were vaccinated on schedules that differed from those tested in clinical trials. Our aim was to assess the effectiveness of the vaccine against various pneumococcal serotypes, and to measure the effectiveness of the recommended dose schedule and of catch-up and incomplete schedules. Methods Invasive disease, defined as isolation of pneumococcus from a sterile site, was identified in children aged 3–59 months through the US Centers for Disease Control and Prevention's Active Bacterial Core surveillance. We tested isolates for serotype and antimicrobial susceptibility. Three controls, matched for age and zip code were selected for each case. We calculated the matched odds ratio for vaccination using conditional logistic regression, controlling for underlying conditions. Vaccine effectiveness was calculated as one minus the adjusted matched odds ratio times 100%. Findings We enrolled 782 cases and 2512 controls. Effectiveness of one or more doses against vaccine serotypes was 96% (95% CI 93–98) in healthy children and 81% (57–92) in those with coexisting disorders. It was 76% (63–85) against infections that were not susceptible to penicillin. Vaccination prevented disease caused by all seven vaccine serotypes, and by vaccine-related serotype 6A. Several schedules were more protective than no vaccination; three infant doses with a booster were more protective against vaccine-type disease than were three infant doses alone (p=0·0323). Interpretation The seven-valent pneumococcal conjugate vaccine prevents invasive disease in both healthy and chronically ill children. The vaccine is effective when used with various non-standard schedules.

Daptomycin is a lipopeptide antibiotic with activity against several important gram-positive bacterial pathogens, including drug-resistant staphylococci and enterococci. Because the mechanism of action of daptomycin is calcium-dependent depolarization of the cell membrane, susceptibility testing requires medium supplemented with a physiological level of calcium. This study assessed two Food and Drug Administration-cleared commercial test devices for determination of daptomycin MICs, Etest and JustOne. A collection of 220 selected isolates, including Staphylococcus aureus , coagulase-negative staphylococci, Enterococcus faecalis , E. faecium , E. avium , E. durans , E. casseliflavus , and E. gallinarum , were tested by both methods. Included in the collection were 22 S. aureus and 14 Enterococcus sp. isolates that were recovered from patients and were nonsusceptible on the basis of the daptomycin MICs. As the reference method for comparison, all isolates were tested by the Clinical and Laboratory Standards Institute broth microdilution method incorporating cation-adjusted Mueller-Hinton broth with 50 μg/ml calcium. Daptomycin MICs agreed, within 1 twofold dilution, for 97% of the isolates by Etest and for 100% by JustOne. However, daptomycin MICs determined by Etest were 1 dilution lower than the reference MICs for 65% of the Enterococcus sp. isolates tested. This resulted in 28.5% very major (VM) errors (4/14) with enterococci (all E. faecium ) but none (0/22) with staphylococci. Use of JustOne yielded MICs that were 1 dilution lower than the reference MICs for 69% of the staphylococci and 25% of the enterococci. This resulted in 13.6% VM errors (3/22) with staphylococci and 14.3% VM errors (2/14) with enterococci. The manufacturer-recommended JustOne inoculum preparation resulted in mean colony counts of only 5 × 10 4 to 1 × 10 5 CFU/ml in the wells of the strip. Increasing the inoculum to 3 × 10 5 to 4 × 10 5 CFU/ml eliminated two of five VM errors upon retesting. No major interpretive errors occurred with either device. In summary, daptomycin MICs generated by the Etest or JustOne method generally agreed within 1 dilution of the reference daptomycin MICs. However, both devices produced slightly lower MICs that resulted in some VM errors.

BACKGROUND Five of seven serotypes in the pneumococcal conjugate vaccine, introduced for infants in the United States in 2000, are responsible for most penicillin-resistant infections. We examined the effect of this vaccine on invasive disease caused by resistant strains. METHODS We used laboratory-based data from Active Bacterial Core surveillance to measure disease caused by antibiotic-nonsusceptible pneumococci from 1996 through 2004. Cases of invasive disease, defined as disease caused by pneumococci isolated from a normally sterile site, were identified in eight surveillance areas. Isolates underwent serotyping and susceptibility testing. RESULTS Rates of invasive disease caused by penicillin-nonsusceptible strains and strains not susceptible to multiple antibiotics peaked in 1999 and decreased by 2004, from 6.3 to 2.7 cases per 100,000 (a decline of 57 percent; 95 percent confidence interval, 55 to 58 percent) and from 4.1 to 1.7 cases per 100,000 (a decline of 59 percent; 95 percent confidence interval, 58 to 60 percent), respectively. Among children under two years of age, disease caused by penicillin-nonsusceptible strains decreased from 70.3 to 13.1 cases per 100,000 (a decline of 81 percent; 95 percent confidence interval, 80 to 82 percent). Among persons 65 years of age or older, disease caused by penicillin-nonsusceptible strains decreased from 16.4 to 8.4 cases per 100,000 (a decline of 49 percent). Rates of resistant disease caused by vaccine serotypes fell 87 percent. An increase was seen in disease caused by serotype 19A, a serotype not included in the vaccine (from 2.0 to 8.3 per 100,000 among children under two years of age). CONCLUSIONS The rate of antibiotic-resistant invasive pneumococcal infections decreased in young children and older persons after the introduction of the conjugate vaccine. There was an increase in infections caused by serotypes not included in the vaccine.

A case of failure of cefepime treatment of a bloodstream infection with AmpC-producing Klebsiella pneumoniae is reported. The failure was attributed to extended-spectrum β-lactamase (ESBL) acquisition by the isolate, possibly during therapy. Problems encountered with ESBL detection in AmpC-producing isolates are discussed.

Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO 2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (≥0.12 μg/ml and ≥0.25 μg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet (B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP . Two strains had reduced quinolone susceptibility due to mutations of gyrA . The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.

Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP , which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis , including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 μg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was ≥8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of ≤2 lacked this mutation. Of 16 sequenced isolates for which the sulfisoxazole MIC was ≥4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.

The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC, ≥8 mg/liter) S. pneumoniae isolates ( n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002. Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 ( P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes. Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups. Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain 23F -1 clone being most frequent (16% of all isolates). Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones. Fifty percent of the isolates were resistant to any three drug classes. Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA , gyrB , parC , and parE genes. In 16% of the isolates, there was evidence of an active efflux mechanism. An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter). Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread.

Penicillin resistance threatens the treatment of pneumococcal infections. We used sentinel hospital surveillance (1978 to 2001) and population-based surveillance (1995 to 2001) in seven states in the Active Bacterial Core surveillance of the Emerging Infections Program Network to document the emergence in the United States of invasive pneumococcal isolates with very-high-level penicillin resistance (MIC ≥ 8 μg/ml). Very-high-level penicillin resistance was first detected in 1995 in multiple pneumococcal serotypes in three regions of the United States. The prevalence increased from 0.56% (14 of 2,507) of isolates in 1995 to 0.87% in 2001 ( P = 0.03), with peaks in 1996 and 2000 associated with epidemics in Georgia and Maryland. For a majority of the strains the MICs of amoxicillin (91%), cefuroxime (100%), and cefotaxime (68%), were ≥8 μg/ml and all were resistant to at least one other drug class. Pneumonia (50%) and bacteremia (36%) were the most common clinical presentations. Factors associated with very highly resistant infections included residence in Tennessee, age of 23F -4 and 35% were related to England 14- 9. After the introduction of the pneumococcal conjugate vaccine, the incidence of highly penicillin resistant infections decreased by 50% among children

In the United States, clinical microbiology laboratories rely upon the National Committee for Clinical Laboratory Standards (NCCLS) for written standards that guide the important elements of antimicrobial susceptibility testing. Whether a laboratory performs a direct version of an NCCLS standard method (e.g., disk diffusion) or uses a commercial device that has been “cleared” by the Food and Drug Administration because it provides results that are essentially equivalent to the NCCLS reference dilution susceptibility method, the NCCLS standards provide important, up-to-date guidance on the most relevant drugs to report on specific organisms, quality control ranges to assure reproducible results, and “breakpoints” to interpret disk diffusion zones or MICs.

Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA ) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLS B ] resistance; usually encoded by ermA or ermC ). MLS B resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLS B resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA , ermC , and msrA . Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC , 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLS B resistance in this study.

Daptomycin MICs at which 50% of isolates were inhibited (MIC 50 s) and MIC 90 s determined by the NCCLS broth microdilution method were both 0.25 μg/ml (range, 0.06 to 2 μg/ml) for 350 pneumococcal isolates. MICs determined by E test strips on commercially prepared Mueller-Hinton sheep blood agars with different calcium contents were 2 to 3 dilutions higher than those determined by strips that contained daptomycin plus calcium. Daptomycin zone diameters varied little on the same media.

In early 2000, a protein-polysaccharide conjugate vaccine targeting seven pneumococcal serotypes was licensed in the United States for use in young children.We examined population-based data from the Active Bacterial Core Surveillance of the Centers for Disease Control and Prevention to evaluate changes in the burden of invasive disease, defined by isolation of Streptococcus pneumoniae from a normally sterile site. Serotyping and susceptibility testing of isolates were performed. We assessed trends using data from seven geographic areas with continuous participation from 1998 through 2001 (population, 16 million).The rate of invasive disease dropped from an average of 24.3 cases per 100,000 persons in 1998 and 1999 to 17.3 per 100,000 in 2001. The largest decline was in children under two years of age. In this group, the rate of disease was 69 percent lower in 2001 than the base-line rate (59.0 cases per 100,000 vs. 188.0 per 100,000, P0.001); the rate of disease caused by vaccine and vaccine-related serotypes declined by 78 percent (P0.001) and 50 percent (P0.001), respectively. Disease rates also fell for adults; as compared with base line, the rate of disease in 2001 was 32 percent lower for adults 20 to 39 years of age (7.6 cases per 100,000 vs. 11.2 per 100,000, P0.001), 8 percent lower for those 40 to 64 years of age (19.7 per 100,000 vs. 21.5 per 100,000, P=0.03), and 18 percent lower for those 65 years of age or more (49.5 per 100,000 vs. 60.1 per 100,000, P0.001). The rate of disease caused by strains that were not susceptible to penicillin was 35 percent lower in 2001 than in 1999 (4.1 cases per 100,000 vs. 6.3 per 100,000, P0.001).The use of the pneumococcal conjugate vaccine is preventing disease in young children, for whom the vaccine is indicated, and may be reducing the rate of disease in adults. The vaccine provides an effective new tool for reducing disease caused by drug-resistant strains.