Blood-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Carbapenemase-Producing Enterobacteriaceae Directly from Positive Blood Culture Broths.

A variant of the modified carbapenem inactivation method (mCIM) was developed to detect carbapenemase activity directly from positive blood culture broths. The method, termed "Blood-mCIM," was evaluated using Bactec blood culture bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) inoculated with 27 different carbapenemase-producing Enterobacteriaceae (CPE) isolates and 34 different non-CPE isolates. The assay was positive for all blood culture broths inoculated with CPE isolates and negative for all blood culture broths inoculated with non-CPE isolates, corresponding to a diagnostic sensitivity and specificity of 100%, respectively. This assay is inexpensive using "off the shelf" reagents, does not require centrifugation or mechanical lysis, and can be readily implemented in any clinical microbiology laboratory. The Blood-mCIM should facilitate expedient administration of antimicrobial therapy targeted toward CPE bloodstream infections and assist infection control and public health surveillance.

EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-ß-Lactamase-Producing Enterobacteriaceae.

The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-ß-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.

High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and MIC Breakpoints Established by the Clinical and Laboratory Standards Institute for Detection of mecA-Mediated Oxacillin Resistance in Staphylococcus schleiferi.

Staphylococcus schleiferi is a beta-hemolytic, coagulase-variable colonizer of small animals that can cause opportunistic infections in humans. In veterinary isolates, the rate of mecA-mediated oxacillin resistance is significant, with reported resistance rates of >39%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and MIC breakpoints for detection of mecA-mediated oxacillin resistance in 52 human and 38 veterinary isolates of S. schleiferi Isolates were tested on multiple brands of commercial media and according to Clinical and Laboratory Standards Institute (CLSI) methods. Zone diameters and MIC values were interpreted using CLSI breakpoints (CLSI, Performance Standards for Antimicrobial Susceptibility Testing. M100-S27, 2017) for Staphylococcus aureus/Staphylococcus lugdunensis, coagulase-negative staphylococci (CoNS), and Staphylococcus pseudintermedius Results were compared to those of mecA PCR. Twenty-nine of 90 (32%) isolates were mecA positive. Oxacillin inhibition zone sizes and MICs interpreted by S. pseudintermedius breakpoints reliably differentiated mecA-positive and mecA-negative isolates, with a categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) for all media. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis and CoNS breakpoints, CA values were 85% and 75%, respectively, and there were 72% and 64% VMEs, respectively, and 0 MEs. For cefoxitin MICs interpreted using S. aureus/S. lugdunensis breakpoints, CA was 81%, and there were 60% VMEs and no MEs. Our data demonstrate that oxacillin DD or MIC testing methods using the current S. pseudintermedius breakpoints reliably identify mecA-mediated oxacillin resistance in S. schleiferi, while cefoxitin DD and MIC testing methods perform poorly.

Trends in rotavirus from 2001 to 2015 in two paediatric hospitals in Atlanta, Georgia.

We compared rotavirus detection patterns before (2001-2006) and after (2008-2015) rotavirus vaccine introduction. We also compared rotavirus detection patterns in odd (2009, 2011, 2013, 2015) and even (2008, 2010, 2012, 2014) years post-vaccine separately. Results of stool rotavirus antigen testing from inpatient, outpatient and emergency department encounters from July 2000 to July 2015 at two paediatric hospital laboratories in Atlanta, Georgia were reviewed. Post-vaccine, rotavirus detection declined (30.2% vs. 13.7% (overall 54.6% decline, P <0.001)), occurred more frequently outside the rotavirus season (19.8% vs. 3.5%; P < 0.001), and was more common among older children (26 vs. 13 median months of age; P < 0.001). During odd years post-vaccine, rotavirus detection was significantly higher than even years (20.2% vs. 6.4%; P < 0.001). Rotavirus detection declined substantially and developed a biennial pattern in the post-vaccine era. The intensity and temporality of rotavirus detection in odd years post-vaccine resembled that observed pre-vaccine, although considerably reduced in magnitude.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli.

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called "mixed genera" (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l'Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer's instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7% of the 965 isolates tested, with 83.8% correct to the species level and 12.8% limited to a genus-level identification. There was no identification for 1.7% of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory.

The identification and/or prediction of the antimicrobial resistance of microorganisms in clinical materials solely by molecular means in the diagnostic microbiology laboratory is not novel. However, the ability to sequence multitudes of bacterial genomes and deliver and interpret the resultant sequence information in near "real-time" is the basis of next-generation sequencing (NGS) technologies. There have been numerous applications and successes of NGS applications in the clinical and public health domain. However, none have, as yet, delivered perhaps the most sought after application, i.e., the generation of microbial sequence data for "real-time" patient management. In this review, we discuss the use of NGS and whole-genome sequencing (WGS) of microorganisms as a logical next step for the routine diagnosis of infection and the prediction of antimicrobial susceptibility in the clinical microbiology laboratory.

Draft Genome Sequence of the Pandoraea apista LMG 16407 Type Strain.

Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF). To aid in understanding the role of P. apista in CF lung disease, we used Illumina MiSeq and nanopore MinION technology to sequence the whole genome of the P. apista LMG 16407(T).