ABSTRACT
BACKGROUND: Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation. METHODS: We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions. RESULTS: The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses. CONCLUSIONS: This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.).
Subject(s)
Cross Infection/microbiology , Disease Reservoirs/microbiology , Gram-Negative Bacterial Infections/microbiology , Sanitary Engineering , Sphingomonas/genetics , Anti-Bacterial Agents/pharmacology , Hospitals, Federal , Humans , Metagenomics , Microbial Sensitivity Tests , National Institutes of Health (U.S.) , Retrospective Studies , Sphingomonas/drug effects , Sphingomonas/isolation & purification , United States , Water Supply , Whole Genome SequencingABSTRACT
Accurate and reproducible antimicrobial susceptibility testing (AST) of polymyxin antibiotics is critical, as these drugs are last-line therapeutic options for the treatment of multidrug-resistant Gram-negative bacterial infections. However, polymyxin AST in the routine laboratory remains challenging. In this study, we evaluated the performance of an automated broth microdilution (BMD) system (Sensititre, ThermoFisher) compared to that of agar dilution (AD) for colistin and polymyxin B AST of 129 Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex clinical isolates. MICs derived from the Sensititre instrument based on two operator comparisons demonstrated overall categorical agreement (CA) of 86% and 89% compared to AD for colistin and 89% and 92% compared to AD for polymyxin B. However, error rates were higher than recommended by CLSI. Manual inspection of microdilution wells revealed microbial growth and skip wells which were erroneously interpreted by the Aris 2X instrument. Using manually interpreted BMD MICs read by two operators increased the overall categorical agreements to 88% and 95% compared to AD for colistin and 92% and 96% compared to AD for polymyxin B. Laboratories choosing to use the Sensititre platform for polymyxin AST should consider manual evaluation of wells as part of their algorithm.
Subject(s)
Colistin , Reading , Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Humans , Microbial Sensitivity Tests , Polymyxin B/pharmacologyABSTRACT
We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69). The percentages of positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% confidence interval [CI], 65.8% to 90.5%) and 99.8% (95% CI, 98.7% to 100%), respectively. The percent positive agreement increased to 90.0% (95% CI, 74.4% to 96.5%) when considering only samples with moderate to high viral load (cycle threshold [CT ] for the NP, ≤34). Pools of five saliva specimens were also evaluated on three platforms, bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche Cobas 6800. The average loss of signal upon pooling was 2 to 3 CT values across the platforms. The sensitivities of detecting a positive specimen in a pool compared with testing individually were 94%, 90%, and 94% for the CDC 2019-nCoV real-time RT-PCR, Panther Fusion SARS-CoV-2 assay, and Cobas SARS-CoV-2 test, respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport medium for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Mass Screening/methods , SARS-CoV-2/isolation & purification , Saliva/virology , Humans , Nasopharynx , Reverse Transcriptase Polymerase Chain Reaction , Specimen Handling/methodsABSTRACT
Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products that fail release criteria (because of potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies. We developed a systematic protocol for the assessment of positive microbial sterility test results using retrospective data from 2007 to 2016. This protocol was validated and applied prospectively between October 2016 and September 2017 to 13 products from which positive sterility results had been reported. Viable and nonviable environmental monitoring (EM) data were collected concurrently as part of a facility control assessment. Three of 13 (23%) positive sterility results were attributable to bone marrow collections that had been contaminated with skin flora during harvest; all were infused without pertinent infectious sequelae. Of the remaining 10, 1 was deemed a true positive and was discarded before infusion, whereas 9 were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and 6 were withheld because of patient issues unrelated to microbial sterility results. No postinfusion-associated infectious complications were documented. Almost half of the positive EM findings were skin flora. Paired detection of an organism in both product and associated EM was identified in 1 case. Application of our validated protocol to positive product sterility test results allowed for systematic data compilation for regulatory evaluation and provided comprehensive information to clinical investigators to ensure timely and strategic management for product recipients.
Subject(s)
Blood Cells , Cell- and Tissue-Based Therapy , Disinfection , Quality Control , Blood Cells/microbiology , Blood Cells/virology , Humans , Retrospective StudiesABSTRACT
The emergence of cell therapy programs in large academic centers has led to an increasing demand for clinical laboratories to assist with product sterility testing. Automated blood culture systems have shown promise as alternatives to the manual USP<71> compendial method, but current published data are limited by small organism test sets, particularly for molds. In 2015, failure of the Bactec FX system to detect mold contamination in two products prompted us to evaluate three test systems (compendial USP<71>, Bactec FX, and BacT/Alert Dual-T) over seven different culture combinations, using 118 challenge organisms representative of the NIH current good manufacturing practice (cGMP) environment. At <96 h and <144 h for bacterial and fungal detection, respectively, the compendial USP<71> method significantly outperformed the Bactec FX system (84.7% versus 64.4%; P = 0.0006) but not the BacT/Alert system at 32.5°C (78.8%; P = 0.3116). Extended incubation to 360 h with terminal visual inspection improved sensitivity, without a significant difference between compendial USP<71> and BacT/Alert testing (95.7% versus 89.0%; P = 0.0860); both systems were better than the Bactec FX system (71.2%; P < 0.0001 and P = 0.0003, respectively). The Bactec FX and BacT/Alert systems performed equivalently for 30 isolates derived from clinical bloodstream infections, confirming system optimization for clinical organisms rather than environmental contaminants. Paired Sabouraud dextrose agar (SDA) plates were always positive for fungi within the acceptable time frame. This study shows that the Bactec FX system is suboptimal for product sterility testing, and it provides strong data to support the use of BacT/Alert testing at 32.5°C paired with a supplemental SDA plate as an acceptable alternative to the compendial USP<71> method for product sterility testing.
Subject(s)
Cell- and Tissue-Based Therapy/adverse effects , Drug Contamination , Drug-Related Side Effects and Adverse Reactions/prevention & control , Microbiological Techniques/methods , Cell- and Tissue-Based Therapy/methods , Sensitivity and SpecificityABSTRACT
Diagnostics and research analyses involving samples containing maximum-containment viruses present unique challenges, and inactivation protocols compatible with downstream testing are needed. Our aim was to identify a validated viral inactivation protocol compatible with bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We assessed a panel of bacteria with 6 validated maximum-containment virus-inactivation protocols and report that inactivation with TRIzol or γ-irradiation is compatible with MALDI-TOF MS. The availability, simplicity, and rapidity of TRIzol inactivation make this method the more suitable choice.
Subject(s)
Bacteria/radiation effects , Coinfection/virology , Virus Inactivation/radiation effects , Viruses/radiation effects , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Carbapenemases have become a significant mechanism for broad-spectrum ß-lactam resistance in Enterobacteriaceae and other Gram-negative bacteria such as Pseudomonas and Acinetobacter spp. Intestinal carriage of carbapenemase-producing organisms (CPOs) is an important source of transmission. Isolation of carriers is one strategy that can be used to limit the spread of these bacteria. In this review, we critically examine the clinical performance, advantages, and disadvantages of methods available for the detection of intestinal carriage of CPOs. Culture-based methods (Centers for Disease Control and Prevention [CDC] protocols, chromogenic media, specialized agars, and double-disk synergy tests) for detecting carriage of CPOs are convenient due to their ready availability and low cost, but their limited sensitivity and long turnaround time may not always be optimal for infection control practices. Contemporary nucleic acid amplification techniques (NAATs) such as real-time PCR, hybridization assays, loop-mediated isothermal amplification (LAMP), or a combined culture and NAAT approach may provide fast results and/or added sensitivity and specificity compared with culture-based methods. Infection control practitioners and clinical microbiologists should be aware of the strengths and limitations of available methods to determine the most suitable approach for their medical facility to fit their infection control needs.
Subject(s)
Bacterial Proteins/metabolism , Carrier State/epidemiology , Enterobacteriaceae/enzymology , Epidemiological Monitoring , Gastrointestinal Tract/microbiology , Gram-Negative Bacterial Infections/epidemiology , beta-Lactamases/metabolism , Acinetobacter/enzymology , Acinetobacter/isolation & purification , Bacterial Proteins/genetics , Bacteriological Techniques/methods , Carrier State/microbiology , Cross Infection/prevention & control , Enterobacteriaceae/isolation & purification , Gram-Negative Bacterial Infections/microbiology , Humans , Molecular Diagnostic Techniques/methods , Pseudomonas/enzymology , Pseudomonas/isolation & purification , beta-Lactamases/geneticsABSTRACT
The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Biological Transport/genetics , Biological Transport/physiology , Genes, MDR/genetics , Genes, MDR/physiology , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nucleosides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicityABSTRACT
Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a reference Klebsiella pneumoniae isolate demonstrated â¼99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and K. pneumoniae isolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.
Subject(s)
Drug Resistance, Bacterial , Escherichia coli/genetics , Genes, Bacterial , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests/methods , Plasmids , Sequence Analysis, DNA/methods , Bacterial Infections/diagnosis , Bacterial Infections/microbiology , Escherichia coli/isolation & purification , Klebsiella pneumoniae/isolation & purification , Nanopores , Time Factors , WorkflowABSTRACT
BACKGROUND: Hypothermia in neonatal intensive care unit patients is associated with morbidity. Perioperative normothermia is the standard of care. AIMS: We hypothesized that a quality improvement intervention (transport protocol, transport education, ongoing monitoring) would decrease the incidence of perioperative hypothermia. Secondarily, we hypothesized that patients undergoing surgery at a postmenstrual age of <37 weeks or at a weight of <1.5 kg would be at higher risk for perioperative hypothermia. METHODS: Lean Six Sigma methodology was used to institute a quality improvement intervention. In a retrospective chart review, we identified 708 cases for which the neonatal intensive care unit was the preoperative and postoperative destination and documented patient characteristics, including postoperative temperature. Cardiac surgical cases and cases with no postoperative temperature record were excluded. RESULTS: Patients in the postintervention group had a statistically significant decrease in hypothermia compared to those in the preintervention group (P < 0.001; OR: 0.17; 95% CI: 0.09-0.31). The absolute risk of hypothermia was 23% in the preintervention group and 6% in the postintervention group. Weight <1.5 kg on day of surgery (P = 0.45; OR: 0.63; 95% CI: 0.16-2.24) and postmenstrual age (P = 0.91; OR: 1.07; 95% CI: 0.33-3.98) were not risk factors. Odds of hypothermia were increased in patients undergoing interventional cardiology procedures (P = 0.003; OR: 17.77; 95% CI: 2.07-125.7). CONCLUSIONS: Perioperative hypothermia is a challenge in the care of neonatal intensive care unit patients; however, a thermoregulation intervention can decrease the incidence with sustained results. Future studies can examine why certain procedures have a tendency toward increased perioperative hypothermia, determine the relative value of quality improvement interventions, and characterize the morbidity and mortality associated with perioperative hypothermia in neonatal intensive care unit patients.
Subject(s)
Body Temperature , Critical Care/methods , Hypothermia/prevention & control , Intensive Care Units, Neonatal , Intraoperative Complications/prevention & control , Perioperative Care/methods , Postoperative Complications/prevention & control , Female , Humans , Infant, Newborn , Male , Retrospective Studies , Risk FactorsABSTRACT
Multidrug-resistant bacteria are responsible for substantial morbidity and mortality worldwide. Tracking the nosocomial spread of resistant bacteria is critical to infection control. Mellmann et al. (J. Clin. Microbiol. 54:2874-2881, 2016, http://dx.doi.org/10.1128/JCM.00790-16) have described prospective whole-genome sequencing with core genome multilocus sequencing typing (cgMLST) analysis for real-time surveillance and have addressed the practical aspects of implementing this type of operation in the hospital setting.
Subject(s)
Cross Infection/microbiology , Genome, Bacterial/genetics , Infection Control/methods , Molecular Epidemiology/methods , Multilocus Sequence Typing/methods , Drug Resistance, Multiple, Bacterial/genetics , Enterococcus faecium/drug effects , Enterococcus faecium/genetics , Enterococcus faecium/isolation & purification , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/isolation & purification , Germany , Hospitals , Humans , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Vancomycin-Resistant Enterococci/genetics , Vancomycin-Resistant Enterococci/isolation & purificationABSTRACT
Rapid detection of blaKPC-containing organisms can significantly impact infection control and clinical practices, as well as therapeutic choices. Current molecular and phenotypic methods to detect these organisms, however, require additional testing beyond routine organism identification. In this study, we evaluated the clinical performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to detect pKpQIL_p019 (p019)-an â¼11,109-Da protein associated with certain blaKPC-containing plasmids that was previously shown to successfully track a clonal outbreak of blaKPC-pKpQIL-Klebsiella pneumoniae in a proof-of-principle study (A. F. Lau, H. Wang, R. A. Weingarten, S. K. Drake, A. F. Suffredini, M. K. Garfield, Y. Chen, M. Gucek, J. H. Youn, F. Stock, H. Tso, J. DeLeo, J. J. Cimino, K. M. Frank, and J. P. Dekker, J Clin Microbiol 52:2804-2812, 2014, http://dx.doi.org/10.1128/JCM.00694-14). PCR for the p019 gene was used as the reference method. Here, blind analysis of 140 characterized Enterobacteriaceae isolates using two protein extraction methods (plate extraction and tube extraction) and two peak detection methods (manual and automated) showed sensitivities and specificities ranging from 96% to 100% and from 95% to 100%, respectively (2,520 spectra analyzed). Feasible laboratory implementation methods (plate extraction and automated analysis) demonstrated 96% sensitivity and 99% specificity. All p019-positive isolates (n = 26) contained blaKPC and were carbapenem resistant. Retrospective analysis of an additional 720 clinical Enterobacteriaceae spectra found an â¼11,109-Da signal in nine spectra (1.3%), including seven from p019-containing, carbapenem-resistant isolates (positive predictive value [PPV], 78%). Instrument tuning had a significant effect on assay sensitivity, highlighting important factors that must be considered as MALDI-TOF MS moves into applications beyond microbial identification. Using a large blind clinical data set, we have shown that spectra acquired for routine organism identification can also be analyzed automatically in real time at high throughput, at no additional expense to the laboratory, to enable rapid detection of potentially blaKPC-containing carbapenem-resistant isolates, providing early and clinically actionable results.
Subject(s)
Bacteriological Techniques/methods , Enterobacteriaceae/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Lactamases/analysis , Carbapenems/pharmacology , Enterobacteriaceae/chemistry , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/microbiology , Humans , Plasmids/analysis , Prospective Studies , Retrospective Studies , Sensitivity and Specificity , beta-Lactam ResistanceABSTRACT
This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.
Subject(s)
Bacteriological Techniques/methods , Nocardia Infections/diagnosis , Nocardia Infections/microbiology , Nocardia/classification , Nocardia/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Nocardia/chemistry , Reproducibility of Results , Sensitivity and Specificity , United StatesABSTRACT
Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.
Subject(s)
Aeromonas hydrophila/enzymology , Aeromonas hydrophila/genetics , Anal Canal/microbiology , Bacterial Proteins/genetics , Genome, Bacterial , Sequence Analysis, DNA , beta-Lactamases/genetics , Adult , Aeromonas hydrophila/classification , Aeromonas hydrophila/isolation & purification , Epidemiological Monitoring , Feces/microbiology , Genetic Variation , Genotype , Humans , Molecular EpidemiologyABSTRACT
BACKGROUND: Commensal Gram-negative (CGN) microbiota have been identified on human skin by DNA sequencing; however, methods to reliably culture viable Gram-negative skin organisms have not been previously described. RESULTS: Through the use of selective antibiotics and minimal media we developed methods to culture CGN from skin swabs. We identified several previously uncharacterized CGN at the species level by optimizing growth conditions and limiting the inhibitory effects of nutrient shock, temperature, and bacterial competition, factors that may have previously limited CGN isolation from skin cultures. CONCLUSIONS: Our protocol will permit future functional studies on the influences of CGN on skin homeostasis and disease.
Subject(s)
Bacteriological Techniques/methods , Gram-Negative Bacteria/growth & development , Skin/microbiology , Culture Media , Gram-Negative Bacteria/isolation & purification , Humans , MicrobiotaABSTRACT
We evaluated the clinical performance of Check-Direct CPE for carbapenemase detection directly from 301 perirectal swabs (258 patients) in a nonoutbreak setting. Culture of a PCR-confirmed, carbapenemase-containing organism, or history of colonization with such organism within the previous 2 weeks, was used as the reference standard. Check-Direct CPE demonstrated a sensitivity value, specificity value, positive predictive value (PPV), and negative predictive value (NPV) of 100% (all bla(KPC)), 88%, 21%, and 100%, respectively. False positives accounted for 79% (n = 34) of samples for which a cycle threshold (C(T)) value was reached. Simulated studies to evaluate specimen pooling as an approach to minimize costs showed no difference in C(T) values for pooled groups of three or five that each contained a single specimen spiked with â¼1,500 CFU bla(KPC) Klebsiella pneumoniae; however, the detection rate dropped to 60% at a seeded concentration of â¼150 CFU. When data were pooled, C(T) values for bla(KPC) were higher for heavy-feces-containing than for light-feces-containing liquid-suspended specimens. Furthermore, C(T) values for liquid-suspended specimens were 4 to 5 C(T) values lower (i.e., represented greater sensitivity) than those seen in direct swab analysis. Culture was equivalent to or better than Check-Direct CPE for 13/15 (87%) isolates tested in a limit-of-detection analysis. Detection of a carbapenemase gene at a C(T) cutoff value of ≤35 was culture confirmed in 23/24 (96%) of cases; however, C(T) values of >35 overlapped broadly between culture-positive (n = 21) and culture-negative (n = 36) specimens. Check-Direct CPE will likely prove most useful in high-prevalence areas or in outbreak settings where rapid carbapenemase detection is critical for infection control management.
Subject(s)
Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/enzymology , Genotyping Techniques/methods , Microbial Sensitivity Tests/methods , Multiplex Polymerase Chain Reaction/methods , beta-Lactam Resistance , beta-Lactamases/analysis , Automation, Laboratory/methods , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Humans , Predictive Value of Tests , Sensitivity and Specificity , beta-Lactamases/geneticsABSTRACT
Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla(KPC) carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An â¼ 11,109-Da MS peak corresponding to a gene product of the bla(KPC) pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla(KPC)-negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla(KPC) Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla(KPC) Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.
Subject(s)
Bacterial Typing Techniques/methods , Disease Outbreaks , Enterobacteriaceae Infections/epidemiology , Enterobacteriaceae/classification , Plasmids/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Lactam Resistance , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Carbapenems/pharmacology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enterobacteriaceae/chemistry , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Enterobacteriaceae Infections/microbiology , Genes, Bacterial , Humans , Molecular Epidemiology/methods , Molecular Weight , Sequence Analysis, DNA , Time FactorsABSTRACT
The prevalence of obesity has increased dramatically worldwide and has become a critical public health priority. Obesity is associated with many co-morbid conditions, including hypertension, diabetes, and cardiovascular disease. Although the physiology of obesity is complex, a healthy diet and sufficient exercise are two elements known to be critical to combating this condition. Years of research on the Mediterranean diet, which is high in fresh fruits and vegetables, nuts, fish, and olive oil, have demonstrated a reduction in numerous non-communicable chronic diseases associated with this diet. There is strong evidence to support an anti-inflammatory effect of the diet, and inflammation is a key driver of obesity. Changes in diet alter the gut microbiota which are intricately intertwined with human physiology, as gut microbiota-derived metabolites play a key role in biological pathways throughout the body. This review will summarize recent published studies that examine the potential role of gut metabolites, including short-chain fatty acids, bile acids, trimethylamine-N-oxide, and lipopolysaccharide, in modulating inflammation after consumption of a Mediterranean-like diet. These metabolites modulate pathways of inflammation through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, toll-like receptor 4 signaling, and macrophage driven effects in adipocytes, among other mechanisms.
ABSTRACT
Ultra-processed foods (UPFs) are foods that are industrially processed and are often pre-packaged, convenient, energy-dense, and nutrient-poor. UPFs are widespread in the current Western diet and their proposed contribution to non-communicable diseases such as obesity and cardiovascular disease is supported by numerous studies. UPFs are hypothesized to affect the body in multiple ways, including by inducing changes in the gut microbiome. This review summarizes the available research on the effect of UPFs on the gut microbiome. We also review current usage of the NOVA food classification system in randomized controlled trials and observational studies and how its implementation effects UPF research. Despite some differences in methodology between studies, results often associate UPF consumption with a number of negative health consequences. There are attempts to standardize a UPF classification system; however, reaching and implementing a consensus is difficult. Future studies focusing on the mechanisms by which UPFs effect the body, including through the microbiome and metabolome, will be essential to refine our understanding of the effects of UPFs on human health.
Subject(s)
Fast Foods , Gastrointestinal Microbiome , Humans , Gastrointestinal Microbiome/physiology , Fast Foods/microbiology , Food Handling , Nutritive Value , Food, ProcessedABSTRACT
Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease.