The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.

The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3's activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7.

Nosocomial Outbreak of Drug-Resistant Streptococcus pneumoniae Serotype 9V in an Adult Respiratory Medicine Ward.

Streptococcus pneumoniae infections arising in hospitalized patients are often assumed to be sporadic and linked to community acquisition. Here, whole-genome sequencing was used to demonstrate nosocomial acquisition of antimicrobial-resistant sequence type 156 (ST156) serotype 9V S. pneumoniae in 3 respiratory patients that resulted in two bacteremias and one lower respiratory tract infection. Two of the cases arose in patients who had recently been discharged from the hospital and were readmitted from the community. Nosocomial spread was suspected solely because of the highly unusual resistance pattern and case presentations within 24 h of one another. The outbreak highlights the potential for rapid transmission and the short incubation period in the respiratory ward setting.

The impact of enhanced cleaning within the intensive care unit on contamination of the near-patient environment with hospital pathogens: a randomized crossover study in critical care units in two hospitals.

OBJECTIVES: To determine the effect of enhanced cleaning of the near-patient environment on the isolation of hospital pathogens from the bed area and staff hands. DESIGN: Prospective randomized crossover study over the course of 1 yr. SETTING: Intensive care units at two teaching hospitals. PATIENTS: There were 1252 patients staying during enhanced cleaning and 1331 staying during standard cleaning. INTERVENTIONS: In each of six 2-month periods, one unit was randomly selected for additional twice-daily enhanced cleaning of hand contact surfaces. MEASUREMENTS AND MAIN RESULTS: Agar contact samples were taken at five sites around randomly selected bed areas, from staff hands, and from communal sites three times daily for 12 bed days per week. Patients admitted in the year commencing April 2007 were analyzed for hospital-acquired colonization and infection. Over the course of 1152 bed days, 20,736 samples were collected. Detection of environmental methicillin-resistant Staphylococcus aureus per bed-area day was reduced during enhanced cleaning phases from 82 of 561 (14.6%) to 51 of 559 (9.1%) (adjusted odds ratio, 0.59; 95% confidence interval, 0.40-0.86; p = .006). Other targeted pathogens (Acinetobacter baumannii, extended-spectrum ß-lactamase-producing Gram-negative bacteria, vancomycin-resistant enterococci, and Clostridium difficile) were rarely detected. Subgroup analyses showed reduced methicillin-resistant Staphylococcus aureus contamination on doctors' hands during enhanced cleaning (3 of 425; 0.7% vs. 11 of 423; 2.6%; adjusted odds ratio, 0.26; 95% confidence interval, 0.07-0.95; p = .025) and a trend to reduction on nurses' hands (16 of 1647; 1.0% vs. 28 of 1694; 1.7%; adjusted odds ratio 0.56; 95% confidence interval, 0.29-1.08; p = .077). All 1252 critical care patients staying during enhanced and 1,331 during standard cleaning were included, but no significant effect on patient methicillin-resistant Staphylococcus aureus acquisition was observed (adjusted odds ratio, 0.98; 95% confidence interval, 0.58-1.65; p = .93). CONCLUSIONS: Enhanced cleaning reduced environmental contamination and hand carriage, but no significant effect was observed on patient acquisition of methicillin-resistant Staphylococcus aureus. TRIAL REGISTRY: ISRCTN. Identifier: 06298448. http://www.controlled-trials.com/isrctn/.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

The Sialidase NanS Enhances Non-TcsL Mediated Cytotoxicity of Clostridium sordellii.

The clostridia produce an arsenal of toxins to facilitate their survival within the host environment. TcsL is one of two major toxins produced by Clostridium sordellii, a human and animal pathogen, and is essential for disease pathogenesis of this bacterium. C. sordellii produces many other toxins, but the role that they play in disease is not known, although previous work has suggested that the sialidase enzyme NanS may be involved in the characteristic leukemoid reaction that occurs during severe disease. In this study we investigated the role of NanS in C. sordellii disease pathogenesis. We constructed a nanS mutant and showed that NanS is the only sialidase produced from C. sordellii strain ATCC9714 since sialidase activity could not be detected from the nanS mutant. Complementation with the wild-type gene restored sialidase production to the nanS mutant strain. Cytotoxicity assays using sialidase-enriched culture supernatants applied to gut (Caco2), vaginal (VK2), and cervical cell lines (End1/E6E7 and Ect1/E6E7) showed that NanS was not cytotoxic to these cells. However, the cytotoxic capacity of a toxin-enriched supernatant to the vaginal and cervical cell lines was substantially enhanced in the presence of NanS. TcsL was not the mediator of the observed cytotoxicity since supernatants harvested from a TcsL-deficient strain displayed similar cytotoxicity levels to TcsL-containing supernatants. This study suggests that NanS works synergistically with an unknown toxin or toxins to exacerbate C. sordellii-mediated tissue damage in the host.

The misuse and overuse of non-sterile gloves: application of an audit tool to define the problem.

BACKGROUND: The use of non-sterile gloves (NSG) has become routine in the delivery of health care, often for procedures for which they are not required; their use may increase the risk of cross contamination and is generally not integrated into hand hygiene audit. This paper describes a small-scale application and validation of an observational audit tool devised to identify inappropriate use of NSG and potential for cross contamination. METHODS: Two observers simultaneously observed the use of NSG during episodes of care in an acute hospital setting. The inter-rater reliability (IRR) of the audit tool was measured corrected for chance agreement using Kappa. RESULTS: A total of 22 episodes of care using NSG were observed. In 68.6% (24/35) of procedures there was no contact with blood/body fluid; in 54.3% (19/35) NSG-use was inappropriate. The IRR was 100% for eight of 12 components of the tool. For hand hygiene before and after NSG removal it was 82% (Kappa = 0.72) and 95% (Kappa = 0.87). CONCLUSIONS: In this small-scale application of a glove-use audit tool we demonstrated over-use and misuse of NSG and potential for cross transmission on gloved hands. The audit tool provides an effective mechanism for integrating glove use into the audit of hand hygiene behaviour.

Bowel management in the intensive care unit.

The area of bowel care in the intensive care unit (ICU) is often overlooked in the holistic care of the critically ill individual. With the primary concern of optimising patients to preserve life the problem of bowel care has been given less priority. The guidelines included within this service improvement paper offer a simple approach to bowel care management with the use of an algorithm and visual display score to be used in conjunction with the algorithm. This was developed in the intensive care unit of the Royal Free Hospital, London and is presently in use.

The use of adenosine triphosphate bioluminescence to assess the efficacy of a modified cleaning program implemented within an intensive care setting.

BACKGROUND: A total environmental cleaning system based on microfiber technology was implemented within 2 intensive care units (ICUs). The efficacy of this modified cleaning program was assessed using adenosine triphosphate (ATP) bioluminescence. METHODS: A team of trained hygiene technicians cleaned all near-patient furniture and equipment twice a day using ultramicrofiber cloths. Every week for 40 weeks, 10 surfaces within a randomly selected bed area were sampled using the 3M Clean-Trace Clinical Hygiene Monitoring System (3M Health Care Ltd, Loughborough, United Kingdom). The ability of the modified cleaning program to reduce surface contamination to "acceptable" levels was measured against previously proposed benchmark ATP values. RESULTS: In comparison with normal cleaning procedures routinely carried out by the nurses, the modified cleaning program significantly reduced (P < .001) the ATP readings obtained from surfaces within the near-patient environment. In both ICUs, 95% of surfaces sampled after modified cleaning had relative light unit values of <500 and were deemed "clean." Almost 90% of the surfaces could also be "passed" using the more stringent benchmark value of 250 relative light units. However, regardless of benchmark value used, the majority of surfaces sampled could also be considered adequately clean prior to them being cleaned by the hygiene technicians. CONCLUSION: The use of ATP bioluminescence has been proposed as a means to improve the management of hospital cleaning. Use of benchmark values can help continually monitor the efficacy of existing cleaning programs. However, when evaluating novel or new cleaning practices, baseline cleanliness (ie, the level of cleanliness routinely achieved using normal cleaning procedures) must also be taken into consideration, or the efficacy of modified cleaning will be overestimated.