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The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Diagnosis of ventilator-assisted pneumonia (VAP) requires pathogen quantitation of respiratory samples. Current quantitative culture methods require overnight growth, and pathogen identification requires an additional step. Automated microscopy can perform rapid simultaneous identification and quantitation of live, surface-immobilized bacteria extracted directly from patient specimens using image data collected over 3 h. Automated microscopy was compared to 1 μL loop culture and standard identification methods for Staphylococcus aureus and Pseudomonas spp. in 53 remnant bronchoalveolar lavage specimens. Microscopy identified 9/9 S. aureus and 7/7 P. aeruginosa in all specimens with content above the VAP diagnostic threshold. Concordance for specimens containing targets above the diagnostic threshold was 13/16, with concordance for sub-diagnostic content of 86/90. Results demonstrated that automated microscopy had higher precision than 1 μL loop culture (range ~0.55 log versus ≥1 log), with a dynamic range of ~4 logs (~10(3) to 10(6) CFU/mL)., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Population analysis was performed for 42 Escherichia coli isolates to determine whether heterogeneity of resistance was a factor in piperacillin-tazobactam category differences between agar dilution and broth microdilution. Of 20 isolates discordant between methods, 80% were heterogeneous. Of 22 isolates in agreement, 59% were homogeneous. Heterogeneity and homogeneity rates for those in agreement were significantly different from those that were discordant (P value, 0.010). Heterogeneity of resistance expression appears to be an important factor in category differences observed between broth microdilution and agar dilution for piperacillin-tazobactam.

Unlabelled: A positive blood culture is a critical result that requires prompt identification of the causative agent. This article describes a simple method to identify microorganisms from positive blood culture broth within the time taken to perform a Gram stain (<20 min). The method is based on intrinsic fluorescence spectroscopy (IFS) of whole cells and required development of a selective lysis buffer, aqueous density cushion, optical microcentrifuge tube, and reference database. A total of 1,121 monomicrobial-positive broth samples from 751 strains were analyzed to build a database representing 37 of the most commonly encountered species in bloodstream infections or present as contaminants. A multistage algorithm correctly classified 99.6% of unknown samples to the Gram level, 99.3% to the family level, and 96.5% to the species level. There were no incorrect results given at the Gram or family classification levels, while 0.8% of results were discordant at the species level. In 8/9 incorrect species results, the misidentified isolate was assigned to a species of the same genus. This unique combination of selective lysis, density centrifugation, and IFS can rapidly identify the most common microbial species present in positive blood cultures. Faster identification of the etiologic agent may benefit the clinical management of sepsis. Further evaluation is now warranted to determine the performance of the method using clinical blood culture specimens., Importance: Physicians often require the identity of the infective agent in order to make life-saving adjustments to empirical therapy or to switch to less expensive and/or more targeted antimicrobials. However, standard identification procedures take up to 2 days after a blood culture is signaled positive, and even most rapid molecular techniques take several hours to provide a result. Other techniques are faster (e.g., matrix-assisted laser desorption ionization-time of flight [MALDI-TOF] mass spectrometry) but require time-consuming manual processing steps and expensive equipment. There remains a clear need for a simple, inexpensive method to rapidly identify microorganisms directly from positive blood cultures. The promising new method described in this research article can identify microorganisms in minutes by optical spectroscopy, thus permitting the lab to simultaneously report the presence of a positive blood culture and the organism's identity.

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

Antimicrobial resistance has emerged as one of the most-significant health care problems of the new millennium, and the clinical microbiology laboratory plays a central role in optimizing the therapeutic management of patients with infection. This minireview explores the potential value of innovative methods for antimicrobial susceptibility testing of microorganisms that could provide valuable alternatives to existing methodologies in the very near future.

Background: Toll-like receptors (TLR) can initiate various immune responses and are therefore activated under diverse infectious states. Previous studies have focused on TLR3 primarily as an antiviral pathway. However, recent research has demonstrated its efficacy in bacterial infection. Having developed a murine double injury model of cecal ligation and puncture (CLP) followed by Pseudomonas aeruginosa (Pa), we hypothesized that targeted administration of Poly I:C, a TLR3 agonist, would protect mice against secondary pneumonia., Material and Methods: B6 mice underwent CLP followed 4 d afterward by an intranasal dose of Pa. Animals were given Poly I:C or vehicle (phosphate-buffered saline) intranasally 24 h post CLP and every day thereafter for a total of 6 d. For acute studies, mice were sacrificed at two time points, 4 d post CLP and 1 d post pneumonia (Pa)., Results: Poly I:C treatment led to a significant improvement in survival (69% versus 33%). Cytokine analysis from bronchioalveolar lavage displayed significant differences both immediately before and after pneumonia. Bronchioalveolar lavage cultures taken at 24 h post double injury showed significantly higher colony counts in the lungs of control animals compared with those of Poly I:C animals. Measurements of TLR3 expression showed significant increases within both the immune and lung epithelial cells of Poly I:C-treated mice. Finally, the lungs of treated animals had significant increases in lymphocytes and innate cells., Conclusions: The prophylactic treatment applied in this clinically relevant model further illustrates the overarching hypothesis of immune dysfunction and the possibility of corrective immune modulation within the setting of sepsis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Capillary-based PCR ribotyping was used to quantify the presence/absence and relative abundance of 98 Clostridium difficile ribotypes from clinical cases of disease at health care institutions in six states of the United States. Regionally important ribotypes were identified, and institutions in close proximity did not necessarily share more ribotype diversity than institutions that were farther apart.

We describe a novel, semiautomated Clostridium difficile typing platform that is based on PCR-ribotyping in conjunction with a semiautomated molecular typing system. The platform is reproducible with minimal intra- or interassay variability. This method exhibited a discriminatory index of 0.954 and is therefore comparable to more arduous typing systems, such as pulsed-field gel electrophoresis.

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.

The endogenous bacteria have been hypothesized to play a significant role in the pathophysiology of critical illness, although their role in sepsis is poorly understood. The purpose of this study was to determine how commensal bacteria alter the host response to sepsis. Conventional and germ-free (GF) C57Bl/6 mice were subjected to Pseudomonas aeruginosa pneumonia. All GF mice died within 2 days, whereas 44% of conventional mice survived for 7 days (P = 0.001). Diluting the dose of bacteria 10-fold in GF mice led to similar survival in GF and conventional mice. When animals with similar mortality were assayed for intestinal integrity, GF mice had lower levels of intestinal epithelial apoptosis but similar levels of proliferation and intestinal permeability. Germ-free mice had significantly lower levels of tumor necrosis factor and interleukin 1β in bronchoalveolar lavage fluid compared with conventional mice without changes in systemic cytokine production. Under conventional conditions, sepsis unmasks lymphocyte control of intestinal epithelial apoptosis, because sepsis induces a greater increase in gut apoptosis in Rag-1 mice than in wild-type mice. However, in a separate set of experiments, gut apoptosis was similar between septic GF Rag-1 mice and septic GF wild-type mice. These data demonstrate that the endogenous bacteria play a protective role in mediating mortality from pneumonia-induced sepsis, potentially mediated through altered intestinal apoptosis and the local proinflammatory response. In addition, sepsis-induced lymphocyte-dependent increases in gut epithelial apoptosis appear to be mediated by the endogenous bacteria.

Background: Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis., Methods: Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded., Results: IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival., Conclusions: The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.

The identification and/or prediction of the antimicrobial resistance of microorganisms in clinical materials solely by molecular means in the diagnostic microbiology laboratory is not novel. However, the ability to sequence multitudes of bacterial genomes and deliver and interpret the resultant sequence information in near "real-time" is the basis of next-generation sequencing (NGS) technologies. There have been numerous applications and successes of NGS applications in the clinical and public health domain. However, none have, as yet, delivered perhaps the most sought after application, i.e., the generation of microbial sequence data for "real-time" patient management. In this review, we discuss the use of NGS and whole-genome sequencing (WGS) of microorganisms as a logical next step for the routine diagnosis of infection and the prediction of antimicrobial susceptibility in the clinical microbiology laboratory.

Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

Background: Optimal treatment regimens for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are not well-defined., Objectives: This study describes the treatment and outcomes in patients with urinary tract infection (UTI) caused by KPC-producing Enterobacteriaceae., Methods: This retrospective cohort study analyzed data from adult inpatients with bacteriuria caused by KPC-positive organisms treated at Barnes-Jewish Hospital from June 1, 2006, to February 1, 2008. KPC-positive isolates were identified utilizing disk-diffusion susceptibility testing and confirmed to contain bla(KPC) via molecular methods., Results: Twenty-one patients met the inclusion criteria and all were classified as having symptomatic UTI. The majority of patients were female (15/21 [71%]), and the mean (SD) age was 62.4 (15.2) years. Successful clinical and microbiologic responses were observed in 16 patients (76%) for both outcomes. Patients with urinary catheters had them removed or replaced in 9 of 15 cases (60%). Antibiotics active against the isolated pathogen were provided in 14 of 21 cases (67%), often after considerable delay (median, 72.5 hours [range, 4-312 hours]). All 7 patients receiving aminoglycoside therapy had successful clinical and microbiologic responses, and in vitro testing of an extended antibiotic panel revealed high susceptibility rates for tigecycline (28/29 [97%]), minocycline (22/29 [76%]), and fosfomycin (25/29 [86%]) against the KPC-positive isolates., Conclusions: Although receipt of appropriate therapy was delayed in many cases, clinical outcomes investigated revealed [corrected] high rates of successful response in this defined group of patients Aminoglycosides and tetracycline derivatives suggested therapeutic promise in the treatment of KPC-producing Enterobacteriaceae UTI., (Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.)

Background: The small intestine plays a crucial role in the pathophysiology of sepsis and has been referred to as the "motor" of the systemic inflammatory response. One proposed mechanism is that toxic gut-derived lipid factors, transported in mesenteric lymph, induce systemic injury and distant organ failure. However, the pathways involved are yet to be defined and the role of intestinal chylomicron assembly and secretion in transporting these lipid factors is unknown. Here we studied the outcome of sepsis in mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO), which exhibit a block in chylomicron assembly together with lipid malabsorption., Methodology/principal Findings: Mttp-IKO mice and controls underwent intratracheal injection with either Pseudomonas aeruginosa or sterile saline. Mttp-IKO mice exhibited decreased seven-day mortality, with 0/20 (0%) dying compared to 5/17 (29%) control mice (p<0.05). This survival advantage in Mttp-IKO mice, however, was not associated with improvements in pulmonary bacterial clearance or neutrophil infiltration. Rather, Mttp-IKO mice exhibited protection against sepsis-associated decreases in villus length and intestinal proliferation and were also protected against increased intestinal apoptosis, both central features in control septic mice. Serum IL-6 levels, a major predictor of mortality in human and mouse models of sepsis, were elevated 8-fold in septic control mice but remained unaltered in septic Mttp-IKO mice. Serum high density lipoprotein (HDL) levels were reduced in septic control mice but were increased in septic Mttp-IKO mice. The decreased levels of HDL were associated with decreased hepatic expression of apolipoprotein A1 in septic control mice., Conclusions/significance: These studies suggest that strategies directed at blocking intestinal chylomicron secretion may attenuate the progression and improve the outcome of sepsis through effects mediated by metabolic and physiological adaptations in both intestinal and hepatic lipid flux.

Mortality in the intensive care unit frequently results from the synergistic effect of two temporally distinct infections. This study examined the pathophysiology of a new model of intra-abdominal sepsis followed by methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Mice underwent cecal ligation and puncture (CLP) or sham laparotomy followed 3 days later by an intratracheal injection of MRSA or saline. Both CLP/saline and sham/MRSA mice had 100% survival, whereas animals with CLP followed by MRSA pneumonia had 67% 7-day survival. Animals subjected to CLP/MRSA had increased bronchoalveolar lavage concentrations of MRSA compared with sham/MRSA animals. Animals subjected to sham/MRSA pneumonia had increased bronchoalveolar lavage levels of interleukin 6 (IL-6), tumor necrosis factor α, and granulocyte colony-stimulating factor compared with those given intratracheal saline, whereas CLP/MRSA mice had a blunted local inflammatory response with markedly decreased cytokine levels. Similarly, animals subjected to CLP/saline had increased peritoneal lavage levels of IL-6 and IL-1β compared with those subjected to sham laparotomy, whereas this response was blunted in CLP/MRSA mice. Systemic cytokines were upregulated in both CLP/saline and sham/MRSA mice, and this was blunted by the combination of CLP/MRSA. In contrast, no synergistic effect on pneumonia severity, white blood cell count, or lymphocyte apoptosis was identified in CLP/MRSA mice compared with animals with either insult in isolation. These results indicate that a clinically relevant model of CLP followed by MRSA pneumonia causes higher mortality than could have been predicted from studying either infection in isolation, and this was associated with a blunted local (pulmonary and peritoneal) and systemic inflammatory response and decreased ability to clear infection.

Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the host's susceptibility to nosocomial infections and Candida albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mortality from pneumonia is mediated, in part, through extrapulmonary causes. Epidermal growth factor (EGF) has broad cytoprotective effects, including potent restorative properties in the injured intestine. The purpose of this study was to determine the efficacy of EGF treatment following Pseudomonas aeruginosa pneumonia. FVB/N mice underwent intratracheal injection of either P. aeruginosa or saline and were then randomized to receive either systemic EGF or vehicle beginning immediately or 24 h after the onset of pneumonia. Systemic EGF decreased 7-day mortality from 65% to 10% when initiated immediately after the onset of pneumonia and to 27% when initiated 24 h after the onset of pneumonia. Even though injury in pneumonia is initiated in the lungs, the survival advantage conferred by EGF was not associated with improvements in pulmonary pathology. In contrast, EGF prevented intestinal injury by reversing pneumonia-induced increases in intestinal epithelial apoptosis and decreases in intestinal proliferation and villus length. Systemic cytokines and kidney and liver function were unaffected by EGF therapy, although EGF decreased pneumonia-induced splenocyte apoptosis. To determine whether the intestine was sufficient to account for extrapulmonary effects induced by EGF, a separate set of experiments was done using transgenic mice with enterocyte-specific overexpression of EGF (IFABP-EGF [intestinal fatty acid-binding protein linked to mouse EGF] mice), which were compared with wild-type mice subjected to pneumonia. IFABP-EGF mice had improved survival compared with wild-type mice following pneumonia (50% vs. 28%, respectively, P < 0.05) and were protected from pneumonia-induced intestinal injury. Thus, EGF may be a potential adjunctive therapy for pneumonia, mediated in part by its effects on the intestine.

Background: Gram-negative bacteria are an important cause of severe sepsis. Recent studies have demonstrated reduced susceptibility of Gram-negative bacteria to currently available antimicrobial agents., Methods: We performed a retrospective cohort study of patients with severe sepsis who were bacteremic with Pseudomonas aeruginosa, Acinetobacter species, or Enterobacteriaceae from 2002 to 2007. Patients were identified by the hospital informatics database and pertinent clinical data (demographics, baseline severity of illness, source of bacteremia, and therapy) were retrieved from electronic medical records. All patients were treated with antimicrobial agents within 12 hours of having blood cultures drawn that were subsequently positive for bacterial pathogens. The primary outcome was hospital mortality., Results: A total of 535 patients with severe sepsis and Gram-negative bacteremia were identified. Hospital mortality was 43.6%, and 82 (15.3%) patients were treated with an antimicrobial regimen to which the causative pathogen was resistant. Patients infected with a resistant pathogen had significantly greater risk of hospital mortality (63.4% vs 40.0%; P < 0.001). In a multivariate analysis, infection with a pathogen that was resistant to the empiric antibiotic regimen, increasing APACHE II scores, infection with Pseudomonas aeruginosa, healthcare-associated hospital-onset infection, mechanical ventilation, and use of vasopressors were independently associated with hospital mortality., Conclusions: In severe sepsis attributed to Gram-negative bacteremia, initial treatment with an antibiotic regimen to which the causative pathogen is resistant was associated with increased hospital mortality. This finding suggests that rapid determination of bacterial susceptibility could influence treatment choices in patients with severe sepsis potentially improving their clinical outcomes., (Copyright © 2011 Society of Hospital Medicine.)

Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later, after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was associated with increases in Th1 cytokines TNF-α and IFN-γ in bronchoalveolar lavage fluid and decreased production of the Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed in Bim(-/-) mice. Septic Bim(-/-) mice with cancer also had increased mortality compared with septic WT mice with cancer. These data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia.

Objective: To determine whether exposure to antimicrobial agents in the previous 90 days resulted in decreased bacterial susceptibility and increased hospital mortality in patients with severe sepsis or septic shock attributed to Gram-negative bacteremia., Design: A retrospective cohort study of hospitalized patients (January 2002 to December 2007)., Setting: Barnes-Jewish Hospital, a 1200-bed urban teaching hospital., Patients: Seven hundred fifty-four consecutive patients with Gram-negative bacteremia complicated by severe sepsis or septic shock., Interventions: Data abstraction from computerized medical records., Measurements and Main Results: Escherichia coli (30.8%), Klebsiella pneumoniae (23.2%), and Pseudomonas aeruginosa (17.6%) were the most common isolates from blood cultures. Three hundred ten patients (41.1%) had recent antibiotic exposure. Cefepime was the most common agent with previous exposure (50.0%) followed by ciprofloxacin (32.6%) and imipenem or meropenem (28.7%). Patients with prior antibiotic exposure had significantly higher rates of resistance to cefepime (29.0% vs. 7.0%), piperacillin/tazobactam (31.9% vs. 11.5%), carbapenems (20.0% vs. 2.5%), ciprofloxacin (39.7% vs. 17.6%), and gentamicin (26.1% vs. 7.9%) (p < .001 for all comparisons). Patients with recent antibiotic exposure had greater inappropriate initial antimicrobial therapy (45.4% vs. 21.2%; p < .001) and hospital mortality (51.3% vs. 34.0%; p < .001) compared with patients without recent antibiotic exposure. Multivariate logistic regression analysis demonstrated that recent antibiotic exposure was independently associated with hospital mortality (adjusted odds ratio, 1.70; 95% confidence interval, 1.41-2.06; p = .005). Other variables independently associated with hospital mortality included use of vasopressors, infection resulting from P. aeruginosa, inappropriate initial antimicrobial therapy, increasing Acute Physiology and Chronic Health Evaluation II scores, and the number of acquired organ failures., Conclusions: Recent antibiotic exposure is associated with increased hospital mortality in Gram-negative bacteremia complicated by severe sepsis or septic shock. Clinicians caring for patients with severe sepsis or septic shock should consider recent antibiotic exposure when formulating empiric antimicrobial regimens for suspected Gram-negative bacterial infection.

Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays.

The intestinal microbiota is important for induction of inflammatory bowel disease (IBD). IBD is associated with complex shifts in microbiota composition, but it is unclear whether specific bacterial subsets induce IBD and, if so, whether their proportions in the microbiota are altered during disease. Here, we fulfilled Koch's postulates in host-genotype-specific fashion using a mouse model of IBD with human-relevant disease-susceptibility mutations. From screening experiments we isolated common commensal Bacteroides species, introduced them into antibiotic-pretreated mice, and quantitatively reisolated them in culture. The bacteria colonized IBD-susceptible and -nonsusceptible mice equivalently, but induced disease exclusively in susceptible animals. Conversely, commensal Enterobacteriaceae were >100-fold enriched during spontaneous disease, but an Enterobacteriaceae isolate failed to induce disease in antibiotic-pretreated mice despite robust colonization. We thus demonstrate that IBD-associated microbiota alterations do not necessarily reflect underlying disease etiology. These findings establish important experimental criteria and a conceptual framework for understanding microbial contributions to IBD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We describe a case of a young woman who had methicillin-resistant Staphylococcus aureus USA300 clone (MRSA-USA300)-associated Lemierre's syndrome and secondary necrotizing pneumonia and cerebral infarcts. We also review 11 cases of S. aureus-associated Lemierre's syndrome reported in the literature from 1965 to 2010. Recognition of S. aureus as an emergent cause of Lemierre's syndrome informs the initial empirical antibiotic choice for this life-threatening condition and may positively impact patient outcomes.

The natural history of contemporary Staphylococcus aureus nasal colonization was evaluated in community children during a 1-year period. Methicillin-susceptible S. aureus nasal carriage was more persistent than methicillin-resistant S. aureus nasal carriage, which was usually self-limited. Children with persistent staphylococcal colonization often carried identical strains. Identification of persistent methicillin-resistant S. aureus carriers might inform strategies for decolonization and reduction of staphylococcal transmission.

In this study, we tested the abilities of the Vitek 2, BD Phoenix, and Kirby Bauer disk diffusion tests to detect carbapenemase production in a collection of 14 Klebsiella pneumoniae carbapenemase (KPC)-producing non-Klebsiella pneumoniae isolates. In addition, we evaluated 13 KPC-positive K. pneumoniae isolates by using each of these methods and applied both 2009 and 2010 CLSI carbapenem interpretive guidelines.

Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h.

We compared ChromID Strepto B agar (STRB; bioMérieux, Inc.), a selective and differential medium for group B streptococcus, with culture using neomycin-nalidixic acid agar (NNA) and LIM broth. STRB alone was more sensitive (87.7%) than NNA alone (79.0%), while each had a sensitivity of 100% when used in conjunction with LIM broth.

Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had approximately 40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-gamma) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-gamma production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.

BBL CHROMagar VanRE (CVRE) was compared with bile esculin azide agar plus vancomycin to screen for vancomycin-resistant enterococcus (VRE) colonization. CVRE distinguishes Enterococcus faecalis (green colonies) from Enterococcus faecium (mauve colonies) on the basis of chromogenic substrate use. CVRE sensitivity and specificity were 98.6% and 99.1%. Positive and negative predictive values were 95.9% and 99.7%.

In January 2006, the Clinical and Laboratory Standards Institute (CLSI) updated breakpoints for vancomycin susceptibility testing for Staphylococcus aureus such that an MIC greater than 2 mg/liter was considered to represent nonsusceptibility to vancomycin. Previously, an MIC of 4 mg/liter had been considered to represent susceptibility. Additionally, in 2009, the CLSI altered the guidelines for staphylococci such that disk diffusion was no longer an acceptable means for testing vancomycin susceptibility in these organisms. To accommodate the change in breakpoints and methodological updates, we designed a medium consisting of brain heart infusion agar with 3 mg/liter vancomycin (BHI-V3) to screen for isolates of S. aureus with reduced susceptibility to vancomycin. We challenged this medium using a previously characterized collection of 100 isolates of S. aureus, including 55 vancomycin-susceptible isolates and 45 isolates representing vancomycin-intermediate strains of S. aureus (VISA) (with vancomycin MICs of 4 mg/liter or greater). All of the VISA isolates grew on the agar, for 100% sensitivity. Nineteen vancomycin-susceptible isolates also grew on the agar, for 65% specificity. We then incorporated BHI-V3 into clinical practice. In the first 60 days postimplementation, we identified 17 potential VISA isolates out of 421 S. aureus isolates tested. Thirteen out of the 17 were confirmed to represent VISA isolates. In light of the excellent sensitivity of this medium, we recommend that clinical laboratories incorporate BHI-V3 to screen for vancomycin-nonsusceptible isolates of S. aureus.

Objective: Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals., Design: Prospective, randomized controlled study., Setting: Animal laboratory in a university medical center., Subjects: C57Bl/6 mice., Interventions: Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival., Measurements and Main Results: Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice., Conclusions: When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.

Objective: Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities., Design: Prospective, randomized controlled study., Setting: Animal laboratory in a university medical center., Interventions: Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points., Measurements and Main Results: The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs., Conclusions: Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.

Three reference MIC methods approved by the Clinical and Laboratory Standards Institute were compared by testing 567 staphylococci against trimethoprim-sulfamethoxazole. Category agreement ranged from 94.9% (broth macrodilution versus broth microdilution) to 98.6% (agar dilution versus broth microdilution). Twenty-seven strains resistant by broth macrodilution were susceptible by broth microdilution.

The performance of the VersaTREK blood culture system for the detection of a bacterial contamination of platelet concentrates was assessed using samples spiked with serial dilutions of nine bacterial species associated with platelet contamination. The system detected growth for all organisms in <20 h, and the detection sensitivity was <20 CFU/ml.

During 6 months, we obtained Enterobacteriaceae isolates from patients with gram-negative bacteremia at a 1,250-bed teaching hospital in St. Louis, MO, and compared carbapenem susceptibilities with the presence of bla(KPC), a transferable carbapenemase gene. Three (1.2%) out of 243 isolates were bla(KPC) positive. Ertapenem nonsusceptibility had a low positive predictive value.

Background: Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare., Materials and Methods: We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia., Results: Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight., Conclusions: These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

Bloodstream infections are an important cause of morbidity and mortality. Physician orders for blood cultures often specify that blood specimens be collected at or around the time of a temperature elevation, presumably as a means of enhancing the likelihood of detecting significant bacteremia. In a multicenter study, which utilized retrospective patient chart reviews as a means of collecting data, we evaluated the timing of blood culture collection in relation to temperature elevations in 1,436 patients with bacteremia and fungemia. The likelihood of documenting bloodstream infections was not significantly enhanced by collecting blood specimens for culture at the time that patients experienced temperature spikes. A subset analysis based on patient age, gender, white blood cell count and specific cause of bacteremia generally also failed to reveal any associations.

Background: Diagnosis of acute infection in the critically ill remains a challenge. We hypothesized that circulating leukocyte transcriptional profiles can be used to monitor the host response to and recovery from infection complicating critical illness., Methodology/principal Findings: A translational research approach was employed. Fifteen mice underwent intratracheal injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 27 mechanically ventilated patients every two days for up to three weeks. Significant heterogeneity of VAP microarray profiles was observed secondary to patient ethnicity, age, and gender, yet 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP. Principal components analysis of these 85 genes appeared to differentiate between the responses of subjects who did versus those who did not develop VAP, as defined by a general trajectory (riboleukogram) for the onset and resolution of VAP. As patients recovered from critical illness complicated by acute infection, the riboleukograms converged, consistent with an immune attractor., Conclusions/significance: Here we present the culmination of a mouse pneumonia study, demonstrating for the first time that disease trajectories derived from microarray expression profiles can be used to quantitatively track the clinical course of acute disease and identify a state of immune recovery. These data suggest that the onset of an infection-specific transcriptional program may precede the clinical diagnosis of pneumonia in patients. Moreover, riboleukograms may help explain variance in the host response due to differences in ethnic background, gender, and pathogen. Prospective clinical trials are indicated to validate our results and test the clinical utility of riboleukograms.

Lymphocyte apoptosis is a hallmark of sepsis and contributes to disease mortality. In other acute injuries, such as myocardial and cerebral ischemia/reperfusion, apoptosis plays a significant role in disease-associated morbidity and mortality. We previously showed that constitutive activation of the potent antiapoptotic Akt/protein kinase B signaling pathway in lymphocytes both reduces sepsis-induced lymphocyte apoptosis and confers a significant survival advantage compared to wild-type littermates. Here, we demonstrate a therapeutic approach to acutely augment Akt activity in a wild-type animal. A cell-permeable peptide conjugated to the Akt-binding domain of the endogenous Akt coactivator, Tcl-1, prolongs Akt activity, activates extracellular regulated kinase (ERK) signaling and protects lymphocytes from numerous apoptotic stimuli both in vitro and in vivo. Molecular approaches to activate the antiapoptotic Akt and ERK signaling pathways may provide a novel tool to study these signaling pathways, as well as a new antiapoptotic strategy for the treatment of sepsis and other acute injuries.

We compared the performance of a chromogenic agar medium chromID VRE (bioMérieux, Marcy-l'Etoile, France) designed to recover and identify vancomycin-resistant enterococci (VRE) from clinical specimens with bile esculin azide vancomycin (BEAV) agar. For this study, 120 stool specimens were plated on chromID VRE and BEAV and examined after 24 and 48 h. At 24 h, the sensitivity and specificity were as follows: BEAV, 90.2% and 73%, respectively; chromID VRE, 86.3% and 100.0%, respectively. Furthermore, we determined that the sensitivity and specificity of chromID VRE for Enterococcus faecium were 85.4% and 100%, respectively, and for Enterococcus faecalis, 90% and 100%, respectively. We conclude that chromID VRE provides an equivalent sensitivity for the recovery of VRE from stool specimens, with improved specificity, and the added advantage of providing differentiation between vancomycin-resistant E. faecium and E. faecalis.

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.

Pseudomonas aeruginosa is a ubiquitous gram-negative rod that can cause a well-recognized, acquired skin infection from bacterial colonization of contaminated water called "hot tub folliculitis." We report an outbreak of pseudomonas skin infection associated with the use of a hot tub at a pool party in 33 children. In particular, 2 of the children were admitted to our hospital; both presented with high leukocyte counts, intermittent low grade fevers, and painful, erythematous nodules and papules on their palms and soles. One of the 2 children also presented with small erythematous pustular lesions on the face and trunk, which led to the diagnosis. Cultures from these pustules grew P aeruginosa. Thirty two other children at this pool/hot tub party developed similar lesions of varying severity 6 to 48 hours after the party. These findings were most consistent with the diagnosis of pseudomonas folliculitis/hot hand.

Background: Nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) can be a precursor to serious infection, and decolonization with topical mupirocin has been studied as a means of preventing clinical infection. Mupirocin resistance in patients with MRSA has been reported, usually in the context of widespread mupirocin use., Methods: Patients admitted to a surgical intensive care unit (SICU) had nasal swab cultures for MRSA performed at admission, weekly, and at discharge in an active surveillance program. Collected MRSA isolates were tested for mupirocin resistance, and molecular analysis was performed. Clinical data on the characteristics and outcomes of the patients who stayed in the SICU for >48 h were collected prospectively., Results: Of the 302 MRSA isolates available for testing, 13.2% were resistant to mupirocin, with 8.6% having high-level resistance (minimum inhibitory concentration, >or=512 microg/mL) and 4.6% having low-level resistance (minimum inhibitory concentration, 8-256 microg/mL). Patients admitted to the SICU for >48 h who were colonized with mupirocin-resistant MRSA were more likely to have been admitted to our hospital during the previous year (P=.016), were older (P=.009), and had higher in-hospital mortality (16% vs. 33%; P=.027), compared with patients colonized with mupirocin-susceptible MRSA. Molecular analysis of the mupirocin-resistant isolates revealed that 72.5% of isolates contained staphylococcal cassette chromosome mec II. Repetitive sequence polymerase chain reaction typing revealed that high-level mupirocin resistance was present in multiple clonal groups. The rate of mupirocin use hospital-wide during the study period was 6.08 treatment-days per 1000 patient-days., Conclusions: We documented a high rate of mupirocin resistance in MRSA isolates from SICU patients, despite low levels of in-hospital mupirocin use.