Michael J. Noto

Related Clones Containing SCCmec Type IV Predominate among Clinically Significant Staphylococcus epidermidis Isolates

ABSTRACT SCCmec is a mobile genetic element that carries the gene (mecA) mediating methicillin resistance in staphylococci. For Staphylococcus aureus, four SCCmec types have been described, one (type IV) of which has been associated with newly identified community-acquired methicillin-resistant S. aureus. However, the distribution of SCCmec types among S. epidermidis is not known. SCCmec typing of a collection of 44 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered between 1973 and 1983 from the blood of patients with prosthetic valve endocarditis (PVE) was performed by PCR amplification of key genetic elements (mecA, mecI, IS1272, and ccrAB). Of the 44 isolates, 1 (2%) harbored SCCmec type I, 15 (34%) harbored type II, 12 (28%) harbored type III, and 16 (36%) harbored type IV. The complete nucleotide sequence of SCCmec type IV was determined for 16 isolates and found to be identical in size (24 kb) and 98% homologous to DNA sequences published for S. aureus. Type IV SCCmec was also common (5 of 10 isolates) among a geographically dispersed collection of 10 recent (1998 to 2001) S. epidermidis bloodstream isolates. Multilocus sequence typing (MLST) (using the same seven genes presently employed for S. aureus MLST) of these MRSE isolates and of 10 additional recent geographically dispersed methicillin-susceptible isolates demonstrated that all 16 PVE isolates and 2 of 5 recent isolates harboring type IV SCCmec were in three related clonal groups. All three MSSE PVE isolates recovered from patients between 1976 and 1979 were in the same clonal groups as type IV SCCmec MRSE isolates. These data support the hypothesis of intra- and interspecies transfer of type IV SCCmec and suggest that there are clonal associations in S. epidermidis that correlate with SCCmec type.

Chlorhexidine bathing and health care-associated infections: a randomized clinical trial.

IMPORTANCE Daily bathing of critically ill patients with the broad-spectrum, topical antimicrobial agent chlorhexidine is widely performed and may reduce health care-associated infections. OBJECTIVE To determine if daily bathing of critically ill patients with chlorhexidine decreases the incidence of health care-associated infections. DESIGN, SETTING, AND PARTICIPANTS A pragmatic cluster randomized, crossover study of 9340 patients admitted to 5 adult intensive care units of a tertiary medical center in Nashville, Tennessee, from July 2012 through July 2013. INTERVENTIONS Units performed once-daily bathing of all patients with disposable cloths impregnated with 2% chlorhexidine or nonantimicrobial cloths as a control. Bathing treatments were performed for a 10-week period followed by a 2-week washout period during which patients were bathed with nonantimicrobial disposable cloths, before crossover to the alternate bathing treatment for 10 weeks. Each unit crossed over between bathing assignments 3 times during the study. MAIN OUTCOMES AND MEASURES The primary prespecified outcome was a composite of central line-associated bloodstream infections (CLABSIs), catheter-associated urinary tract infections (CAUTIs), ventilator-associated pneumonia (VAP), and Clostridium difficile infections. Secondary outcomes included rates of clinical cultures that tested positive for multidrug-resistant organisms, blood culture contamination, health care-associated bloodstream infections, and rates of the primary outcome by ICU. RESULTS During the chlorhexidine bathing period, 55 infections occurred: 4 CLABSI, 21 CAUTI, 17 VAP, and 13 C difficile. During the control bathing period, 60 infections occurred: 4 CLABSI, 32 CAUTI, 8 VAP, and 16 C difficile. The primary outcome rate was 2.86 per 1000 patient-days during the chlorhexidine and 2.90 per 1000 patient-days during the control bathing periods (rate difference, -0.04; 95% CI, -1.10 to 1.01; P = .95). After adjusting for baseline variables, no difference between groups in the rate of the primary outcome was detected. Chlorhexidine bathing did not change rates of infection-related secondary outcomes including hospital-acquired bloodstream infections, blood culture contamination, or clinical cultures yielding multidrug-resistant organisms. In a prespecified subgroup analysis, no difference in the primary outcome was detected in any individual intensive care unit. CONCLUSION AND RELEVANCE In this pragmatic trial, daily bathing with chlorhexidine did not reduce the incidence of health care-associated infections including CLABSIs, CAUTIs, VAP, or C difficile. These findings do not support daily bathing of critically ill patients with chlorhexidine. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT02033187.

Randomized Trial of Apneic Oxygenation during Endotracheal Intubation of the Critically Ill

RATIONALE Hypoxemia is common during endotracheal intubation of critically ill patients and may predispose to cardiac arrest and death. Administration of supplemental oxygen during laryngoscopy (apneic oxygenation) may prevent hypoxemia. OBJECTIVES To determine if apneic oxygenation increases the lowest arterial oxygen saturation experienced by patients undergoing endotracheal intubation in the intensive care unit. METHODS This was a randomized, open-label, pragmatic trial in which 150 adults undergoing endotracheal intubation in a medical intensive care unit were randomized to receive 15 L/min of 100% oxygen via high-flow nasal cannula during laryngoscopy (apneic oxygenation) or no supplemental oxygen during laryngoscopy (usual care). The primary outcome was lowest arterial oxygen saturation between induction and 2 minutes after completion of endotracheal intubation. MEASUREMENTS AND MAIN RESULTS Median lowest arterial oxygen saturation was 92% with apneic oxygenation versus 90% with usual care (95% confidence interval for the difference, -1.6 to 7.4%; P = 0.16). There was no difference between apneic oxygenation and usual care in incidence of oxygen saturation less than 90% (44.7 vs. 47.2%; P = 0.87), oxygen saturation less than 80% (15.8 vs. 25.0%; P = 0.22), or decrease in oxygen saturation greater than 3% (53.9 vs. 55.6%; P = 0.87). Duration of mechanical ventilation, intensive care unit length of stay, and in-hospital mortality were similar between study groups. CONCLUSIONS Apneic oxygenation does not seem to increase lowest arterial oxygen saturation during endotracheal intubation of critically ill patients compared with usual care. These findings do not support routine use of apneic oxygenation during endotracheal intubation of critically ill adults. Clinical trial registered with www.clinicaltrials.gov (NCT 02051816).

Gene Acquisition at the Insertion Site for SCCmec, the Genomic Island Conferring Methicillin Resistance in Staphylococcus aureus

Staphylococcus aureus becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA. SCCmec is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCCmec attachment site (attB). In an effort to gain a better understanding of the potential that methicillin-sensitive S. aureus (MSSA) isolates have for acquiring SCCmec, the nucleotide sequences of attB and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding attB varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCCmec elements, suggesting SCCmec elements may have evolved through repeated DNA insertions at this locus. In addition, the attB sequences of the majority of MSSA isolates in this collection differ from the attB sequences of strains for which integrase-mediated SCCmec insertion or excision has been demonstrated, suggesting that some S. aureus isolates may lack the ability to site-specifically integrate SCCmec into their chromosomes.

Spontaneous Deletion of the Methicillin Resistance Determinant, mecA, Partially Compensates for the Fitness Cost Associated with High-Level Vancomycin Resistance in Staphylococcus aureus

ABSTRACT Treatment of infections caused by Staphylococcus aureus is often confounded by the bacteriums ability to develop resistance to chemotherapeutic agents. Methicillin-resistant S. aureus (MRSA) arises through the acquisition of staphylococcal chromosomal cassette mec (SCCmec), a genomic island containing the methicillin resistance determinant, mecA. In contrast, resistance to vancomycin can result from exposure to the drug, a mechanism that is not dependent upon a gene acquisition event. Here we describe three MRSA strains that became resistant to vancomycin during passage in the presence of increasing concentrations of the drug. In each case two derivative strains were isolated, one that had lost mecA and one that retained mecA during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec, while the other two strains (strains 3130VR and VP32) deleted portions of their SCCmec elements in a manner that appeared to involve IS431. Conversion to vancomycin resistance caused a decrease in the growth rate that was partially compensated for by the deletion of mecA. In mixed-culture competition experiments, vancomycin-resistant strains that lacked mecA readily outcompeted their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.

Demonstration of the Involvement of Outer Surface Protein E Coiled Coil Structural Domains and Higher Order Structural Elements in the Binding of Infection-Induced Antibody and the Complement-Regulatory Protein, Factor H

Factor H (fH) is an important regulator of the alternative complement cascade. Several human pathogens have been shown to bind fH to their surface, a process that facilitates immune evasion or cell to cell interaction. Among the pathogens that bind fH are some Borrelia species associated with Lyme disease and relapsing fever. The fH-binding proteins of the Lyme spirochetes form two classes (I and II). In Borrelia burgdorferi B31MI, class I includes the outer surface protein E (OspE) paralogs, L39, N38, and P38, whereas the class II group includes A68 and additional proteins that have not yet been identified. To identify the OspE determinants involved in fH and OspE-targeting infection-induced Ab (iAb) binding, deletion, random, and site-directed mutagenesis of L39 were performed. Mutations in several different regions of L39 abolished fH and or iAb binding, indicating that separable domains and residues of OspE are required for ligand binding. Some of the mutants that lost the ability to bind fH, iAb, or both had only a single amino acid change. Site-directed mutagenesis of three putative coiled coil motifs of OspE revealed that these higher order structures are required for fH binding but not for iAb binding. The data presented within demonstrate that the binding of fH and iAb to the OspE protein is mediated by higher order structures and protein conformation. These studies advance our understanding of fH binding as a virulence mechanism and facilitate ongoing efforts to use fH-binding proteins in the development of microbial vaccines.

The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung.

ABSTRACT Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.

Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis.

MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra‐high speed MALDI‐TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI‐TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 μm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra‐high speed MALDI‐TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (<5 ppm) and resolving power (∼75 000 at m/z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor.

Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection

Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and is an urgent public health concern worldwide. Over the past decade, incidence, severity and costs associated with C. difficile infection (CDI) have increased dramatically. CDI is most commonly initiated by antibiotic-mediated disruption of the gut microbiota; however, non-antibiotic-associated CDI cases are well documented and on the rise. This suggests that unexplored environmental, nutrient and host factors probably influence CDI. Here we show that excess dietary zinc (Zn) substantially alters the gut microbiota and, in turn, reduces the minimum amount of antibiotics needed to confer susceptibility to CDI. In mice colonized with C. difficile, excess dietary Zn severely exacerbated C. difficile–associated disease by increasing toxin activity and altering the host immune response. In addition, we show that the Zn-binding S100 protein calprotectin has antimicrobial effects against C. difficile and is an essential component of the innate immune response to CDI. Taken together, these data suggest that nutrient Zn levels have a key role in determining susceptibility to CDI and severity of disease, and that calprotectin-mediated metal limitation is an important factor in the host immune response to C. difficile.

Randomized Trial of Video Laryngoscopy for Endotracheal Intubation of Critically Ill Adults.

Objective:To evaluate the effect of video laryngoscopy on the rate of endotracheal intubation on first laryngoscopy attempt among critically ill adults. Design:A randomized, parallel-group, pragmatic trial of video compared with direct laryngoscopy for 150 adults undergoing endotracheal intubation by Pulmonary and Critical Care Medicine fellows. Setting:Medical ICU in a tertiary, academic medical center. Patients:Critically ill patients 18 years old or older. Interventions:Patients were randomized 1:1 to video or direct laryngoscopy for the first attempt at endotracheal intubation. Measurements and Main Results:Patients assigned to video (n = 74) and direct (n = 76) laryngoscopy were similar at baseline. Despite better glottic visualization with video laryngoscopy, there was no difference in the primary outcome of intubation on the first laryngoscopy attempt (video 68.9% vs direct 65.8%; p = 0.68) in unadjusted analyses or after adjustment for the operator’s previous experience with the assigned device (odds ratio for video laryngoscopy on intubation on first attempt 2.02; 95% CI, 0.82–5.02, p = 0.12). Secondary outcomes of time to intubation, lowest arterial oxygen saturation, complications, and in-hospital mortality were not different between video and direct laryngoscopy. Conclusions:In critically ill adults undergoing endotracheal intubation, video laryngoscopy improves glottic visualization but does not appear to increase procedural success or decrease complications.