MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here we report a novel strategy for the rapid detection of SARS-CoV-2 based on an enrichment approach exploiting the affinity between the virus and cellulose sulfate ester functional groups, hot acid hydrolysis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Virus samples were enriched using cellulose sulfate ester microcolumns. Virus peptides were prepared using the hot acid aspartate-selective hydrolysis and characterized by MALDI-TOF MS. Collected spectra were processed with a peptide fingerprint algorithm, and searching parameters were optimized for the detection of SARS-CoV-2. These peptides provide high sequence coverage for nucleocapsid (N protein) and allow confident identification of SARS-CoV-2. Peptide markers contributing to the detection were rigorously identified using bottom-up proteomics. The approach demonstrated in this study holds the potential for developing a rapid assay for COVID-19 diagnosis and detecting virus variants from a variety of sources, such as sewage and nasal swabs.

SARS-CoV-2 Supply Shortages and Tuberculosis Diagnostics: Current Issues Requiring Immediate Solutions.

The SARS-CoV-2 pandemic has strained manufacturing capacity worldwide, resulting in significant shortages of laboratory supplies both directly and indirectly. Such shortages include probe-based kits for detection of the Mycobacterium tuberculosis complex from positive liquid broth cultures. These shortages and possible loss of this particular assay have consequences for laboratory testing algorithms and public health in the United States. As there are no FDA-approved, commercially available options that currently exist which could immediately fill this gap, laboratories must identify alternatives and plan for modifying current testing algorithms to accommodate this change.

Multicity outbreak of linezolid-resistant Staphylococcus epidermidis associated with clonal spread of a cfr-containing strain.

We report a multicity outbreak of cfr-containing linezolid-resistant Staphylococcus epidermidis in Ohio. Thirty-nine isolates were obtained from 2 hospitals. Two clones with different mechanisms of linezolid resistance were circulating in hospital A. One of these contained the cfr gene, and the other a ribosomal mutation. The clone containing cfr was identical in both hospitals.

Emergence of linezolid-resistant coagulase-negative Staphylococcus in a cancer centre linked to increased linezolid utilization.

OBJECTIVES: The prevalence of linezolid-resistant coagulase-negative Staphylococcus (CoNS) in the MD Anderson Cancer Center rose from 0.6% in 2007 to 5.5% in 2009. The aim of our study was to analyse the relationship between linezolid use and an outbreak of linezolid-resistant CoNS. PATIENTS AND METHODS: We retrospectively identified 27 infection or colonization events. Eleven isolates were available for supplemental investigation; species identification, clonal relatedness and linezolid resistance mutation analysis. The medical records of the affected patients were reviewed and linezolid utilization data were obtained from the pharmacy. RESULTS: Available isolates were confirmed as clonally related Staphylococcus epidermidis. Partial 23S rRNA gene sequencing found a G2576T mutation in all of the isolates tested. All patients received linezolid within 3 months prior to an event. Patients without a prior hospitalization had a longer time from admission to event; 29 versus 3.5 days (P = 0.002). The outbreak was preceded by a 51% increase in inpatient linezolid utilization and 64% of affected patients belonged to the leukaemia service, which had a utilization rate 3.1 times that of the other services (95% confidence interval: 2.96-3.23). CONCLUSIONS: Increased linezolid utilization preceded the appearance of a linezolid-resistant CoNS clone. Patients probably acquired the clonal strain nosocomially, given the longer time from admission to event among patients with no previous admission to the MD Anderson Cancer Center. Linezolid administration then selected this strain, since all patients received linezolid prior to an event. A linezolid utilization rate of >or=13 defined daily doses/100 patient-days was similar to that reported in two other outbreaks and may be the threshold required to generate an outbreak.

Siderophore receptor-mediated uptake of lactivicin analogues in gram-negative bacteria.

Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.

Pyridone-conjugated monobactam antibiotics with gram-negative activity.

Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.

Successful eradication of a monoclonal strain of Klebsiella pneumoniae during a K. pneumoniae carbapenemase-producing K. pneumoniae outbreak in a surgical intensive care unit in Miami, Florida.

We describe the investigation and control of a Klebsiella pneumoniae carbapenemase-producing K. pneumoniae outbreak in a 20-bed surgical intensive care unit during the period from January 1, 2009 through January 1, 2010. Nine patients were either colonized or infected with a monoclonal strain of K. pneumoniae. The implementation of a bundle of interventions on July 2009 successfully controlled the further horizontal spread of this organism.