Instant Detection of Synthetic Cannabinoids on Physical Matrices, Implemented on a Low-Cost, Ultraportable Device.

Synthetic cannabinoids (SCs) make up a class of novel psychoactive substances (NPS), used predominantly in prisons and homeless communities in the U.K. SCs can have severe side effects, including psychosis, stroke, and seizures, with numerous reported deaths associated with their use. The chemical diversity of SCs presents the major challenge to their detection since approaches relying on specific molecular recognition become outdated almost immediately. Ideally one would have a generic approach to detecting SCs in portable settings. The problem of SC detection is more challenging still because the majority of SCs enter the prison estate adsorbed onto physical matrices such as paper, fabric, or herb materials. That is, regardless of the detection modality used, the necessary extraction step reduces the effectiveness and ability to rapidly screen materials on-site. Herein, we demonstrate a truly instant generic test for SCs, tested against real-world drug seizures. The test is based on two advances. First, we identify a spectrally silent region in the emission spectrum of most physical matrices. Second, the finding that background signals (including from autofluorescence) can be accurately predicted is based on tracking the fraction of absorbed light from the irradiation source. Finally, we demonstrate that the intrinsic fluorescence of a large range of physical substrates can be leveraged to track the presence of other drugs of interest, including the most recent iterations of benzodiazepines and opioids. We demonstrate the implementation of our presumptive test in a portable, pocket-sized device that will find immediate utility in prisons and law enforcement agencies around the world.

Detection, discrimination and quantification of amphetamine, cathinone and nor-ephedrine regioisomers using benchtop 1 H and 19 F nuclear magnetic resonance spectroscopy.

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated 1 H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by 1 H NMR (match score median = 0.9933) and GC-MS (RRt = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative 19 F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative 1 H NMR and quantitative 19 F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample.

Comparative study of the analysis of seized samples by GC-MS, 1H NMR and FT-IR spectroscopy within a Night-Time Economy (NTE) setting.

Rapid analysis of surrendered or seized drug samples provides important intelligence for health (e.g. treatment or harm reduction), and custodial services. Herein, three in-situ techniques, GC-MS, 1H NMR and FT-IR spectroscopy, with searchable libraries, are used to analyse 318 samples qualitatively, using technique specific library-based searches, obtained over the period 24th - 29th August 2019. 259 samples were identified as consisting of a single component, of which cocaine was the most prevalent (n = 158). Median match scores for all three techniques were ≥ 0.84 and showed agreement except for metformin (n = 1), oxandrolone (identified as vitamin K by IR (n = 4)), diazepam (identified as zolpidem by FT-IR (n = 2)) and 2-Br-4,5-DMPEA (n = 1), a structural isomer of 2C-B identified as a polymer of cellulose (cardboard) by FT-IR. 51 samples were found to consist of two or more components, of which 49 were adulterated cocaine samples (45 binary and 4 tertiary samples). GC-MS identified all components present in the 49 adulterated cocaine samples, whereas IR identified only cocaine in 88 % of cases (adulterant only = 12 %). The breakdown for 1H NMR spectroscopy was all components identified (51 %), cocaine only (33 %), adulterant only (10 %), cocaine and one adulterant (tertiary mixtures only, 6 %).

Guilty by dissociation: Part A: Development of a rapid Ultra-High Performance Liquid Chromatography (UHPLC)-MS/MS methodology for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS).

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (tG = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Hitting the Jackpot - development of gas chromatography-mass spectrometry (GC-MS) and other rapid screening methods for the analysis of 18 fentanyl-derived synthetic opioids.

In recent years, the occurrence of synthetic opioid fentanyl and its derivatives has grown significantly in forensic casework. This study presents the synthesis and analysis of 18 fentalogs, selected based on information received from local law enforcement. This study provides colorimetric tests, thin-layer chromatography (TLC) which can potentially be utilized for presumptive screening of the target compounds, as bulk powders or as trace-level adulterants. The fully validated confirmatory GC-MS method (employing SIM mode) allows the identification of the 18 derivatives, five commonly encountered controlled substances and four adulterants, within 20 minutes. The cross-validated method described herein provides a sensitive screening and quantitation method for the illicit (and potentially harmful) components at trace levels (LOD = 0.007-0.822 µg/mL and LOQ = 0.023-2.742 µg/mL respectively). Spectral data [1 H-NMR, 13 C-NMR, 19 F-NMR, FT-IR, and HRMS] and assignments for the synthesized reference materials are also provided in the Supplementary Information for laboratories engaged in the routine analysis of fentanyl and its derivatives.

Rapid Identification of Novel Psychoactive and Other Controlled Substances Using Low-Field 1H NMR Spectroscopy.

An automated approach to the collection of 1H NMR (nuclear magnetic resonance) spectra using a benchtop NMR spectrometer and the subsequent analysis, processing, and elucidation of components present in seized drug samples are reported. An algorithm is developed to compare spectral data to a reference library of over 300 1H NMR spectra, ranking matches by a correlation-based score. A threshold for identification was set at 0.838, below which identification of the component present was deemed unreliable. Using this system, 432 samples were surveyed and validated against contemporaneously acquired GC-MS (gas chromatography-mass spectrometry) data. Following removal of samples which possessed no peaks in the GC-MS trace or in both the 1H NMR spectrum and GC-MS trace, the remaining 416 samples matched in 93% of cases. Thirteen of these samples were binary mixtures. A partial match (one component not identified) was obtained for 6% of samples surveyed whilst only 1% of samples did not match at all.

Enantiospecific Synthesis, Chiral Separation, and Biological Activity of Four Indazole-3-Carboxamide-Type Synthetic Cannabinoid Receptor Agonists and Their Detection in Seized Drug Samples.

Synthetic cannabinoid receptor agonists (SCRAs) have been the largest group of illicit psychoactive substances reported to international monitoring and early warning systems for many years. Carboxamide-type SCRAs are amongst the most prevalent and potent. Enantiospecific synthesis and characterization of four indazole-3-carboxamides, AMB-FUBINACA, AB-FUBINACA, 5F-MDMB-PINACA (5F-ADB), and AB-CHMINACA is reported. The interactions of the compounds with CB1 and CB2 receptors were investigated using a G-protein coupled receptor (GPCR) activation assay based on functional complementation of a split NanoLuc luciferase and EC50 (a measure of potency) and Emax (a measure of efficacy) values determined. All compounds demonstrated higher potency at the CB2 receptor than at the CB1 receptor and (S)-enantiomers had an enhanced potency to both receptors over the (R)-enantiomers. The relative potency of the enantiomers to the CB2 receptor is affected by structural features. The difference was more pronounced for compounds with an amine moiety (AB-FUBINACA and AB-CHMINACA) than those with an ester moiety (AMB-FUBINACA and 5F-MDMB-PINACA). An HPLC method was developed to determine the prevalence of (R)-enantiomers in seized samples. Lux® Amylose-1 [Amylose tris(3,5-dimethylphenylcarbamate)] has the greatest selectivity for the SCRAs with a terminal methyl ester moiety and a Lux® i-Cellulose-5 column for SCRAs with a terminal amide moiety. Optimized isocratic separation methods yielded enantiomer resolution values (Rs) ≥ 1.99. Achiral GC-MS analysis of seized herbal materials (n = 16), found 5F-MDMB-PINACA (<1.0-91.5 mg/g herbal material) and AMB-FUBINACA (15.5-58.5 mg/g herbal material), respectively. EMB-FUBINACA, AMB-CHMICA, 5F-ADB-PINACA isomer 2, and ADB-CHMINACA were also tentatively identified. Analysis using chiral chromatography coupled to photodiode array and quadrupole time of flight mass spectrometry (chiral HPLC-PDA-QToF-MS/MS) confirmed that the (S)-enantiomer predominated in all samples (93.6-99.3% (S)-enantiomer). Small but significant differences in synthesis precursor enantiopurity may provide significant differences between synthesis batches or suppliers and warrants further study. A method to compare potency between samples containing different SCRAs at varying concentrations was developed and applied in this small preliminary study. A 10-fold difference in the "intrinsic" potency of samples in the study was noted. With the known heterogeneity of SCRA infused materials, the approach provides a simplified method for assessing and communicating the risk of their use.

Genotypic Characterization of Methicillin-Resistant Staphylococcus aureus Recovered at Baseline from Phase 3 Pneumonia Clinical Trials for Ceftobiprole.

Baseline methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with nosocomial and community-acquired pneumonia collected during Phase 3 trials for ceftobiprole were characterized. Eighty-four unique isolates from patients enrolled in Europe (50.0%), Asia-Western Pacific region (APAC; 20.2%), North America (19.0%), Latin America (8.3%), and South Africa (2.4%) were included. Antimicrobial susceptibility testing was performed by broth microdilution and isolates screened for Panton-Valentine leukocidin. SCCmec and agr types were determined. Strains were subjected to pulsed-field gel electrophoresis and spa typing. Clonal complexes (CCs) were assigned based on spa and/or multilocus sequence typing. Most isolates were CC5-MRSA-I/II/IV (44.0%; 37/84), followed by CC8-MRSA-IV (22.6%; 19/84) and CC239-MRSA-III (21.4%; 18/84). Other MRSA formed seven clonal clusters. Isolates from North America were associated with USA100, while those from South America belonged to the Cordobes/Chilean CC. A greater clonal diversity was observed in Europe; however, each country had CC5, CC8, or CC239 as prevalent lineages. Isolates from APAC were CC5-MRSA-II (47.1%; 8/17) or CC239-MRSA-III (47.1%; 8/17). Isolates carrying SCCmec I and III had ceftobiprole MIC50 values of 2 µg/ml, while those isolates with SCCmec II and IV had MIC50 values of 1 µg/ml. Ceftobiprole inhibited 96% and 100.0% of the isolates at ≤ 2 and ≤ 4 µg/ml, respectively. These isolates represented common circulating MRSA clones. Ceftobiprole demonstrated in vitro activity with a slight variation of minimum inhibitory concentrations (MICs) according to SCCmec or clonal type.

Noninvasive Streptococcus pneumoniae serotypes recovered from hospitalized adult patients in the United States in 2009 to 2012.

This study was conducted to determine the serotype distribution and trends over time of Streptococcus pneumoniae strains associated with noninvasive infections among adult patients ≥18 years of age in the United States (2009 to 2012). A total of 2,927 S. pneumoniae isolates recovered from patients presenting with respiratory infections and obtained mainly (87.0%) from lower respiratory tract specimens (sputum) were included. The levels of the 7-valent pneumococcal conjugate vaccine (PCV7) serotypes remained stable over the 4-year study period (4.6% to 5.5%; P = 0.953). Overall, 13-valent pneumococcal conjugate vaccine (PCV13) serotypes were identified in 32.7% of samples, declining from 33.7% to 35.5% in 2009 to 2011 to 28.2% in 2012 (P = 0.007), with a significant decrease in the levels of serotypes 7F (P = 0.013) and 6A (P = 0.010). The levels of 19A remained constant (15.8% to 17.1%) during 2009 to 2011, dropping to 12.2% in 2012 (P = 0.089). The prevalence of serotypes associated with the 23-valent pneumococcal polysaccharide vaccine (PPSV23), but not PCV13, remained generally stable; however, the prevalence of serotypes 15B and 15C (15B/15C) increased from 2.7% to 6.3% (P = 0.010). The proportion of nonvaccine serotypes increased gradually during the study period (P = 0.044), particularly for serotype 35B (from 3.6% in 2009 to 8.2% in 2012; P = 0.001). Nonsusceptibility rates for penicillin (susceptible breakpoint, ≤2 µg/ml) and clindamycin against PCV7 serotypes decreased over the period. These results suggest the emergence of indirect effects following introduction of PCV13 for infants and young children; continued surveillance is needed to assess the burden of PCV13 serotypes in the adult population after the implementation of age-based recommendations in the United States.

Serotype distribution and antimicrobial susceptibility of USA Streptococcus pneumoniae isolates collected prior to and post introduction of 13-valent pneumococcal conjugate vaccine.

This study evaluated pneumococci cultured from blood or lower respiratory tract specimens from hospitalized patients in the USA (all age groups) during 2011-2012 (N = 1190) and compared findings with those from a similar study performed in 2008 (N = 694). Isolates were tested for susceptibility by broth microdilution and serotypes determined by cpsB sequencing, supplemented with multiplex PCR and capsular swelling assays. Relative percentages of 7-valent pneumococcal conjugate vaccine (PCV7) types were 6.3 and 4.9% in 2008 and 2011-2012, respectively, and the most common PCV7 serotypes (19F and 6B) comprised only 3.7% and 4.0% of all isolates from both periods, respectively. Thirteen-valent pneumococcal conjugate vaccine (PCV13) serotypes represented 42.9% of isolates in 2008 and 30.1% in the second period, and this decrease was driven by 19A and 7F. Non-PCV13 serogroups/serotypes 23A, 15B/15C, 7C, 8, and 31 increased. Penicillin non-susceptibility rates were 9.6-10.0% and 38.9-42.7% when applying the parenteral (i.e. ≥ 4 µg/mL) and oral breakpoints (i.e. ≥ 0.12 µg/mL), respectively. Ceftaroline was the most potent agent tested based on MIC50 and MIC90 values (≤ 0.015 and 0.12 µg/mL, respectively) for both time periods.

Epidemiology and carbapenem resistance mechanisms of carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries.

OBJECTIVES: To evaluate the genetic relatedness and carbapenem resistance mechanisms among carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries. METHODS: Doripenem-non-susceptible (MIC >2 mg/L) isolates were tested for susceptibility to imipenem, meropenem, doripenem, aztreonam, ceftazidime and cefepime with and without phenyl-arginine-ß-naphthylamide (PAßN) (efflux inhibitor) and/or cloxacillin (AmpC inhibitor). Carbapenemase screening was performed by PCR and sequencing. Expression of chromosomal ampC, mexA, mexC, mexE and mexX was determined by quantitative real-time PCR using P. aeruginosa PAO1 or a group of susceptible isolates as baseline. Clonality was evaluated by PFGE and multilocus sequence typing. RESULTS: Among 529 (25.6% overall) carbapenem-non-susceptible P. aeruginosa, 106 were positive for metallo-ß-lactamase (MßL) genes encoding VIM-2 (76 strains), VIM-4 (14), VIM-1 (7) and VIM-5 (5). IMP-15 and three new MßLs (IMP-33, VIM-36 and VIM-37) were detected in one strain each. An increasing prevalence of MßL producers was noted in 2011 (30.6%) compared with previous years (13.4% and 12.3% in 2009 and 2010, respectively). Isolates displayed high genetic diversity, with 401 unique profiles detected. CC235 and ST111 were detected among MßL-producing clusters. The PAßN/cloxacillin effect ranged from 90.0% to 56.5%/from 1.3% to 21.2%. OprD decrease/loss was the most prevalent intrinsic mechanism and was detected among 94.9% of the P. aeruginosa, followed by AmpC (44.4%) and MexAB-OprM (20.1%). When using the susceptible group of isolates as baseline, MexAB-OprM became as prevalent as OprD decrease/loss. CONCLUSIONS: Increasing MßL prevalence is worrisome in various European countries; however, intrinsic resistance mechanisms in a highly genetically diverse population of carbapenem-non-susceptible P. aeruginosa are probably a matter for greater concern in these countries.

Abundant DNase I-sensitive bacterial DNA in healthy porcine lungs and its implications for the lung microbiome.

Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.

Molecular characterization of vancomycin-resistant Enterococcus spp. clinical isolates recovered from hospitalized patients among several medical institutions in China.

The epidemiology and molecular characteristics of vancomycin-resistant enterococci (VRE) from China deserve further investigation. This study reports on the molecular characterization of 101 unique VRE (96 E. faecium and 5 E. faecalis strains) recovered from diverse samples of 12 hospitals in China. MIC results were obtained by reference broth microdilution methods, and vancomycin resistance and virulence genes were screened by polymerase chain reaction. All strains were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. E. faecalis exhibited vancomycin and teicoplanin MIC results at ≥256 µg/mL and harbored vanA, except for 1 vanB-carrying strain (MIC, 32 and 1 µg/mL, respectively). This strain had a unique PFGE pattern and was associated with ST410 (clonal complex [CC]4). E. faecium displayed vancomycin MIC values of ≥256 µg/mL with variable results for teicoplanin (1-256 µg/mL). One E. faecium had a teicoplanin MIC value of 1 µg/mL and carried a vanB, while the other 2 strains had teicoplanin MIC values of 4 and 8 µg/mL and harbored vanA. E. faecalis strains were susceptible to ampicillin, and all VRE displayed a susceptible phenotype to daptomycin, linezolid, and tigecycline. Four E. faecalis from a particular hospital were grouped within a single PFGE type and were associated with ST470 and ST471 (CC4), which are double- and triple-locus variants of ST410 and ST4, respectively. Overall, E. faecium displayed genetic variability, but clonal dissemination was noted within and among hospitals. All E. faecium belonged to STs associated with CC17, except for 1 strain (ST362; CC362). A total of 77.2% and 29.7% of all strains carried esp and hyl, respectively. In conclusion, these results show that vanA-carrying isolates predominated in strains from China, and E. faecium strains are usually associated with a common and human-adapted lineage (CC17). Unlike the majority of clinical E. faecalis (CC2 and CC9), strains included in this study showed ST profiles similar to ST4 (CC4), which has been associated with human infections in other Asia-Pacific countries.

Molecular epidemiology of Staphylococcus epidermidis clinical isolates from U.S. hospitals.

The epidemiology of Staphylococcus epidermidis in U.S. hospitals remains limited. This study aimed to address the genetic backgrounds of linezolid-susceptible and -resistant S. epidermidis strains (isolated in 2010), including cfr-carrying strains. In addition, the antimicrobial susceptibility profiles and linezolid resistance mechanisms among clonal lineages were assessed. A total of 71 S. epidermidis isolates were selected, and linezolid-resistant strains were screened for cfr and mutations in 23S rRNA, L3, and L4. All isolates were subjected to multilocus sequence typing (MLST), and the results were analyzed by eBURST. Overall, 27 sequence types (STs) were detected, and ST5 (21.1%) and ST2 (16.9%) predominated. The majority (62/71; 87.3%) of STs belonged to clonal complex 2 (CC2), which was mostly comprised of subclusters CC2-II (41/62; 66.1%) and CC2-I (21/62; 33.9%). Other STs were grouped within CC23 or CC32 or were singletons. CC2-I strains were more likely to display a methicillin (95.2% versus 33.3 to 70.7%), a linezolid (47.6% versus 0.0 to 7.3%), or a multidrug (81.0% versus 33.3 to 36.6%) resistance phenotype. Among linezolid-resistant isolates, cfr was noted only within CC2 strains, and it was detected equally in the CC2-I (3/10; 30.0%) and CC2-II (1/3; 33.3%) subclusters. 23S rRNA mutations (G2576 [seven strains] and C2534 [one strain]) were observed only among CC2-I (8/10; 80.0%) isolates. Strains showing a G2576 alteration also had M156 (7/7; 100.0%) and/or H146 (6/7; 85.7%) L3 modifications. This study provides an overview of the S. epidermidis clonal distribution and reports higher resistance rates among CC2-I strains. The results show that cfr may be acquired and expressed by both CC2 main subclusters, while 23S rRNA mutations appeared more often within CC2-I strains. Interestingly, these 23S rRNA mutants also had L3 alterations, which may act synergistically or in a compensatory manner to minimize the fitness cost while providing survival advantages under selective pressure.

Detection of Staphylococcus aureus isolates with heterogeneous intermediate-level resistance to vancomycin in the United States.

The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling-area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 µg/ml) or vancomycin-resistant (MIC ≥ 16 µg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 µg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 µg/ml and 0.1% of isolates with vancomycin MICs of 1 µg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.

Activity of ceftaroline and epidemiologic trends in Staphylococcus aureus isolates collected from 43 medical centers in the United States in 2009.

A Staphylococcus aureus surveillance program was initiated in the United States to examine the in vitro activity of ceftaroline and epidemiologic trends. Susceptibility testing by Clinical and Laboratory Standards Institute broth microdilution was performed on 4,210 clinically significant isolates collected in 2009 from 43 medical centers. All isolates were screened for mecA by PCR and evaluated by pulsed-field gel electrophoresis. Methicillin-resistant S. aureus (MRSA) were analyzed for Panton-Valentine leukocidin (PVL) genes and the staphylococcal cassette chromosome mec (SCCmec) type. All isolates had ceftaroline MICs of ≤2 µg/ml with an MIC(50) of 0.5 and an MIC(90) of 1 µg/ml. The overall resistance rates, expressed as the percentages of isolates that were intermediate and resistant (or nonsusceptible), were as follows: ceftaroline, 1.0%; clindamycin, 30.2% (17.4% MIC ≥ 4 µg/ml; 12.8% inducible); daptomycin, 0.2%; erythromycin, 65.5%; levofloxacin, 39.9%; linezolid, 0.02%; oxacillin, 53.4%; tetracycline, 4.4%; tigecycline, 0%; trimethoprim-sulfamethoxazole, 1.6%; vancomycin, 0%; and high-level mupirocin, 2.2%. The mecA PCR was positive for 53.4% of the isolates. The ceftaroline MIC(90)s were 0.25 µg/ml for methicillin-susceptible S. aureus and 1 µg/ml for MRSA. Among the 2,247 MRSA isolates, 51% were USA300 (96.9% PVL positive, 99.7% SCCmec type IV) and 17% were USA100 (93.4% SCCmec type II). The resistance rates for the 1,137 USA300 MRSA isolates were as follows: erythromycin, 90.9%; levofloxacin, 49.1%; clindamycin, 7.6% (6.2% MIC ≥ 4 µg/ml; 1.4% inducible); tetracycline, 3.3%; trimethoprim-sulfamethoxazole, 0.8%; high-level mupirocin, 2.7%; daptomycin, 0.4%; and ceftaroline and linezolid, 0%. USA300 is the dominant clone causing MRSA infections in the United States. Ceftaroline demonstrated potent in vitro activity against recent S. aureus clinical isolates, including MRSA, daptomycin-nonsusceptible, and linezolid-resistant strains.