First description of the performance of the VITEK MITUBE device used with MALDI-TOF mass spectrometry to achieve identification of gram-negative bacteria directly from positive blood culture broth.

Improving the rapidity of identification of bacteria causing bacteremia continues to be a focus for quality improvement within the discipline of clinical microbiology. This multicenter study was used to obtain In Vitro Diagnostic Regulation Conformite Europeenne (CE) mark in Europe and describes the performance of the VITEK MITUBE to matrix-assisted laser desorption/ionization (MALDI) workflow for identification of 10 species of gram-negative bacteria directly from positive blood culture broth without subculture. One hundred twenty-five (125) prospective clinical samples, including (69), (4), (3), (29), (9), (10), and (1), were included; and 123 (98.

assays for clinical isolates of sequence type 131 do not recapitulate infectivity using a murine model of urinary tract infection.

Unlabelled: Sequence type 131 isolates are a major cause of cystitis and pyelonephritis. Many studies rely solely on assays to screen for bacterial virulence factors associated with the pathogenicity of clinical isolates of . Few studies have compared findings with infectivity of clinical isolates.

Molecular epidemiology of β-lactamases in ceftriaxone-resistant Enterobacterales bloodstream infections in the mid-Atlantic United States.

Ceftriaxone-resistant Enterobacterales remain a public health threat; contemporary data investigating their molecular epidemiology are limited. Five hundred consecutive ceftriaxone-resistant (MIC ≥ 4 µg/mL) Enterobacterales bloodstream isolates were collected between 2018 and 2022 from three Maryland hospitals. Broth microdilution confirmed antibiotic susceptibilities.

AmpC induction by imipenem in occurs in the absence of OprD and impacts imipenem/relebactam susceptibility.

In the United States, carbapenem resistance in is linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The β-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility.

What Contributes to the Minimum Inhibitory Concentration? Beyond β-Lactamase Gene Detection in Klebsiella pneumoniae.

Background: Klebsiella pneumoniae is capable of resistance to β-lactam antibiotics through expression of β-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which β-lactamases and outer membrane porins affected β-lactam resistance.

Draft Genome Sequences of the Clinical Isolates Kp 23 and KPM 20.

strains are capable of becoming resistant through multiple mechanisms. Here, we announce draft sequences for Kp 23, a clinical isolate with no plasmid-encoded β-lactamases, and KPM 20, a clinical isolate with no plasmid-encoded β-lactamases and no detectable OmpK35, OmpK36, or PhoE in the outer membrane.

OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC.

Background: Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq.

A Standard Numbering Scheme for Class C β-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser, Lys, Tyr, and Lys), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.

lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

Purpose: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34.

Draft Genome Assemblies of Clinical Isolates of Klebsiella pneumoniae V9011662 and Enterobacter hormaechei Entb306.

and are pathogenic that have been associated with the spread of antibiotic resistance. Here, we report draft genome assemblies of an clinical isolate and a multidrug-resistant clinical isolate of .

Draft Genome Sequence of the Mucoid Clinical Isolate PA34.

is a serious threat to patients suffering from cystic fibrosis. These organisms are exposed to a unique set of selective pressures within the lung. Here, we report the draft genome sequence of a mucoid clinical isolate obtained from a cystic fibrosis patient colonized with .

Impact of CLSI and EUCAST Cefepime breakpoint changes on the susceptibility reporting for Enterobacteriaceae.

Objective: We analyzed the effects of different cefepime MIC breakpoints on Enterobacteriaceae cefepime susceptibility and the presence of AmpC and extended-spectrum β-lactamase (ESBL) genes within the cefepime MIC interpretative categories.

Methods: Using Enterobacteriaceae susceptibility data from 2013 comparisons of MIC breakpoints were performed using Pearson's chi-squared test. Molecular testing on a subset of isolates was done.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 in large sample populations in the absence of sequence analysis.

Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18.

IMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-Å resolution.

Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli.

Objectives: High 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.

Methods: Disc diffusion was used to establish zone size.

The OpdQ porin of Pseudomonas aeruginosa is regulated by environmental signals associated with cystic fibrosis including nitrate-induced regulation involving the NarXL two-component system.

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes chronic infections in immunocompromised hosts. Multiple porins modulate outer membrane permeability under various environmental conditions. The lung environment of cystic fibrosis (CF) patients is unique with changes occurring in nutrient availability, osmolarity, and oxygen content.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay.

Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths.

Whole genome mapping of the first reported case of KPC-2-positive Klebsiella pneumoniae ST258 in Nebraska.

Three ertapenem-resistant Klebsiella pneumoniae carrying bla(KPC-2) were isolated from a single patient in Nebraska over a span of 5 months. A comparative analysis of the genetic relatedness of these isolates was investigated using pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome mapping.

Emergence of carbapenem resistance due to the novel insertion sequence ISPa8 in Pseudomonas aeruginosa.

Chronic lung infections due to the persistence of Pseudomonas aeruginosa in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of P. aeruginosa collected from a patient with cystic fibrosis was challenged with meropenem.

Multiplex high-resolution melting analysis as a diagnostic tool for detection of plasmid-mediated AmpC β-lactamase genes.

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.

Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.

Class B β-lactamases are known as metallo-β-lactamases (MBLs) and they hydrolyze most β-lactams, including carbapenems. IMP-18, an MBL cloned from Pseudomonas aeruginosa, was overexpressed, purified and crystallized by vapour diffusion for X-ray crystallographic analysis. Preliminary X-ray analysis showed that the crystal diffracted to 2.

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours.

Effect of drug treatment options on the mobility and expression of blaKPC.

Objectives: Both transposition and increases in gene expression have been implicated in the success of KPC-producing pathogens, but the stimulus required for these phenomena are unknown. It is possible that exposure to antimicrobials during patient treatment increases bla(KPC) expression or induces Tn4401 transposition. The purpose of this study was to determine if exposure to carbapenems or other antimicrobial drug classes could stimulate expression of bla(KPC) or the in vitro transposition of Tn4401.