Next-generation DNA sequencing (NGS) has progressed enormously over the past decade, transforming genomic analysis and opening up many new opportunities for applications in clinical microbiology laboratories. The impact of NGS on microbiology has been revolutionary, with new microbial genomic sequences being generated daily, leading to the development of large databases of genomes and gene sequences. The ability to analyze microbial communities without culturing organisms has created the ever-growing field of metagenomics and microbiome analysis and has generated significant new insights into the relation between host and microbe. The medical literature contains many examples of how this new technology can be used for infectious disease diagnostics and pathogen analysis. The implementation of NGS in medical practice has been a slow process due to various challenges such as clinical trials, lack of applicable regulatory guidelines, and the adaptation of the technology to the clinical environment. In April 2015, the American Academy of Microbiology (AAM) convened a colloquium to begin to define these issues, and in this document, we present some of the concepts that were generated from these discussions.

A novel metallo-β-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.

A multiplex, real-time TaqMan assay was designed to identify clinical isolates carrying plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 100% when testing characterized AmpC/non-AmpC-producing isolates and randomly selected clinical isolates. This is a rapid assay that can be performed in a clinical microbiology laboratory.A ntibiotic resistance is a global health crisis. There are at least two approaches to address this problem. One way is the design and use of novel therapeutic drug classes to treat infections caused by resistant pathogens. A second approach is the development and implementation of novel surveillance techniques in which to identify not only the pathogen but the resistance mechanisms employed by these organisms. Implementation of molecular-based surveillance techniques is the most immediate response to this crisis and will increase the speed and accuracy of detecting resistance, which is important for both infection control and therapeutic options in hospital and community settings.The most common Gram-negative resistance mechanism associated with ␤-lactams is the production of inactivating ␤-lactamases, including extended-spectrum ␤-lactamases (ESBLs), plasmid-mediated AmpCs, and the Klebsiella pneumoniae carbapenemases (KPCs) (6,12,13,14). K. pneumoniae, Escherichia coli, and Salmonella spp. are the most common organisms that produce plasmid-mediated AmpCs. Genes encoding AmpCs are derived from the chromosomal ampC genes of various members of the Enterobacteriaceae family, including Enterobacter cloacae and Enterobacter asburiae, Citrobacter freundii, Morganella morganii, Aeromonas sobria, Aeromonas hydrophila, and Hafnia alvei (1,3,4,5,7,8,9,10,14,16).Production of plasmid-mediated AmpCs in Gram-negative organisms is clinically important because of their ability to confer resistance to broad-spectrum penicillins, broad/extended-spectrum cephalosporins, monobactams, and the cephamycins (3,4,5,9,14). In addition, the presence of plasmid-mediated AmpCs can mask the phenotypic detection of ESBLs and KPC-producing organisms, which can hinder surveillance and infection control practices (8,11,13,14,17). An additional concern is that plasmidmediated AmpCs are frequently associated with false susceptibility to the cephalosporins in routine susceptibility testing, which increases the risk of therapeutic failure (14). However, given these concerns, there are no guidelines set forth by the CLSI to help clinical microbiologists identify these types of organisms.Modifications to a previously designed endpoint AmpC multiplex PCR has allowed us to develop a real-time multiplex PCR assay using TaqMan probes for the detection of plasmid-mediated AmpC ␤-lactamase genes, which allows ease of implementation into the clinical laboratory (9). The primers and TaqMan probes used for amplification were designed with Beacon Designer 7 software and presented in Table 1. BLAST analysis using sequences submitted to GenBank was used to evaluate the ability of the primer/probe combinations t...

ObjectivesHigh levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam.MethodsDisc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT–PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives.ResultsmRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2–48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5–15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2–3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm.ConclusionsCTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.

High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively. P lasmid-mediated AmpC ␤-lactamase (pmAmpC) genes are derived from the chromosomal ampC genes from various Gram-negative bacterial species. These genes are divided into six families, including bla MOX , bla FOX , bla CMY-2 , bla DHA , bla ACC , and bla ACT . AmpC ␤-lactamases are associated with resistance to broad-spectrum penicillins, broad-spectrum cephalosporins, monobactams, and cephamycins (1-4). The establishment of standardized procedures for the identification of pmAmpC-producing organisms has yet to occur, thereby allowing this resistance mechanism to spread throughout community and hospital settings or be misidentified as an extended-spectrum ␤-lactamase (1, 2). Therapeutic failure has been associated with the inability to detect a resistant phenotype when the organism produced a pmAmpC (5, 6). AmpC multiplex PCR assays previously designed by our laboratory have been modified into a realtime, multiplex assay that uses high-resolution melting (HRM) analysis to detect all six families of pmAmpC genes in Ͻ2 h (7,8). Using this technology, each ampC gene fragment produces an amplicon with distinct melt characteristics to yield six separated melt curves. Primers used in the assay are listed in Table 1 and annealed with 100% specificity to target gene variants (7,8).DNA was extracted from an overnight culture using the FDAapproved QIAamp DSP DNA minikit (Qiagen, Hilden, Germany). The final reaction mixture volume for the multiplex PCR was 25 l. Each PCR mixture consisted of 1ϫ EpiTect Type-iT HRM PCR buffer and the following primer concentrations: 0.6One microliter of DNA template (10 ng/l) was added to 24 l of the master mix. PCR parameters included an initial denaturation at 95°C for 5 min, followed by three-step cycling conditions for 40 cycles consisting of denaturation at 95°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 10 s. Fluorescence acquisition was programmed to the green channel on the extension step of PCR amplification. When using the Rotor-Gene Q, only 10 reactions could be prepared in one master mix without observing a flat line of fluorescence, and therefore, a minimum of two master mixes had to be prepared for each real-time PCR run. HRM analysis followed the cycling parameters with a temperature range of 80 to 89°C that was increased in 0.1°C increments. ScreenClust analysis was attempted to simplify interpretation of HRM results but was unsuccessful in generating the appropriate clusters (Qiagen, Hilden, Germany) (9-11).All primer sets were evaluated for their ability to anneal to target genes using a previously constructed panel of K12 transformants containing pmAmpC gene fragments and HRM analysis using the conditio...