Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

AIMS: To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. METHODS AND RESULTS: Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. CONCLUSIONS: Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials.

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

AIMS: To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. METHODS AND RESULTS: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. CONCLUSIONS: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. SIGNIFICANCE AND IMPACT OF THE STUDY: Hot, humid air is a potential alternative to conventional chemical decontamination.

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores.

AIMS: To compare physical properties of spores that were produced in broth sporulation media at greater than 10(8) spores ml(-1). METHODS AND RESULTS: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0.68 +/- 0.11 microm wide and 1.21 +/- 0.18 microm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0.48 +/- 0.38 microm(3) and 0.97 +/- 0.07 microm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l(-1) glutamate and antifoam. Spores were 0.95 +/- 0.11 microm wide and 1.31 +/- 0.17 microm long. Spore volumes were 0.78 +/- 0.61 microm(3) and volume-equivalent spherical diameters were 1.14 +/- 0.11 microm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. CONCLUSIONS: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.

Characterization of the Bacillus subtilis spore morphogenetic coat protein CotO.

Bacillus spores are protected by a structurally and biochemically complex protein shell composed of over 50 polypeptide species, called the coat. Coat assembly in Bacillus subtilis serves as a relatively tractable model for the study of the formation of more complex macromolecular structures and organelles. It is also a critical model for the discovery of strategies to decontaminate B. anthracis spores. In B. subtilis, a subset of coat proteins is known to have important roles in assembly. Here we show that the recently identified B. subtilis coat protein CotO (YjbX) has an especially important morphogenetic role. We used electron and atomic force microscopy to show that CotO controls assembly of the coat layers and coat surface topography as well as biochemical and cell-biological analyses to identify coat proteins whose assembly is CotO dependent. cotO spores are defective in germination and partially sensitive to lysozyme. As a whole, these phenotypes resemble those resulting from a mutation in the coat protein gene cotH. Nonetheless, the roles of CotH and CotO and the proteins whose assembly they direct are not identical. Based on fluorescence and electron microscopy, we suggest that CotO resides in the outer coat (although not on the coat surface). We propose that CotO and CotH participate in a late phase of coat assembly. We further speculate that an important role of these proteins is ensuring that polymerization of the outer coat layers occurs in such a manner that contiguous shells, and not unproductive aggregates, are formed.

Two class A high-molecular-weight penicillin-binding proteins of Bacillus subtilis play redundant roles in sporulation.

The four class A penicillin-binding proteins (PBPs) of Bacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamed pbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic delta-lactam and L-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.

Value of molecular epidemiologic analysis in a nosocomial methicillin-resistant Staphylococcus aureus outbreak.

OBJECTIVE: To evaluate two molecular epidemiologic methods used in the analysis of a nosocomial methicillin-resistant Staphylococcus aureus (MRSA) outbreak. DESIGN: Restriction endonuclease analysis of plasmid DNA (REAP) was used in the analysis of 45 MRSA isolates. After termination of the outbreak, isolates were retrospectively analyzed in a blind fashion using the newly described technique of arbitrarily primed polymerase chain reaction (AP-PCR). Molecular analyses were compared with epidemiologic and antimicrobial susceptibility data. SETTING: Tertiary care university hospital. SUBJECTS: Twenty-eight patients and 12 employees infected or colonized with MRSA during a 6-week period. RESULTS: A clonal relationship demonstrated among isolates from burn unit patients and staff was clearly distinguishable from MRSA isolates arising from other hospital wards. The combination of REAP and AP-PCR provided complementary information in several instances. Aggressive measures to isolate infected patients and eradicate colonization from patients and staff terminated the outbreak. CONCLUSIONS: Although traditional epidemiologic methods retain their central role in modern hospital infection control, molecular epidemiologic analysis can significantly enhance the ability of infection control officers to analyze and terminate hospital epidemics. The combination of AP-PCR and REAP may prove to be a particularly informative means of tracking the nosocomial spread of microbial strains and their mobile genetic elements.

The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2.

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.

Reducing occupational exposure to blood in the OR.

1. Many occupational exposures to blood in the operating room can be prevented. Some percutaneous injuries may not be preventable, but their frequency can be reduced by implementing engineering and work practice controls. Work practice controls and personal protective equipment can help eliminate mucocutaneous exposures to blood. 2. Identifying risk factors for intraoperative exposure is a vital step in presurgical and intraoperative assessment to plan infection prevention and control interventions. The decision to use risk reduction strategies should be based on probability and type of exposure anticipated rather than on the index of suspicion for bloodborne infection in the patient. 3. The responsibility for using safety and barrier precautions in the operating room remains that of the health-care worker. Not only the patient, but also the health-care worker need to be protected.

Blood exposure and puncture risks for OR personnel.

1. Although all health-care workers are at risk for exposure to bloodborne organisms, OR personnel are the most intensively exposed to blood. Exposures to blood were noted in up to half of the procedures observed. 2. Risk-reduction strategies include using two pairs of puncture-resistant gloves and face protection for all procedures; wearing impermeable gowns during procedures with heavy blood loss; using surgical instruments and techniques that reduce the chance of percutaneous contacts; and adopting protocols for handling sharps, counting sponges, and cleaning the operating room. 3. Better and more comfortable personal protective equipment is needed. Manufacturers should develop risk-protective and cost-effective barriers that reduce risks for patients of surgical wound infections while reducing the risks for health-care workers of exposures to blood and bloodborne pathogens.

A survey of nurses' knowledge, opinions, and reported uses of the Body Substance Isolation system.

The Body Substance Isolation (BSI) system was implemented at the University of California San Diego Medical Center in May 1987. About 2 years later, an evaluation was done of the long-term effects of BSI education and training on the knowledge, attitudes, and reported behaviors of nursing personnel. In June 1989, a questionnaire was sent to 600 nursing personnel, including all 100 nurses in the 20-bed surgical intensive care unit, all 66 charge nurses, and a random sample (434) of the remaining nursing staff (about 1000). Results from the 190 respondents (a response rate of 32%) indicated an understanding of the two purposes of BSI: (1) to reduce nosocomial infection risks to patients and (2) to reduce health care workers' risks of acquiring infections from patients. Over half of the respondents reported handling more than 11 needles per day and nearly half reported recapping contaminated needles two-handed "sometimes or often." Only 54% of the respondents reported they had received hepatitis B vaccine. Although more than two-thirds of the respondents had worked at the University of California San Diego Medical Center during the entire BSI system training, implementation, and follow-up period, there is still room for improvement in knowledge and use of the system, including issues related to the safe handling of sharps.

Hepatitis A through E--current and future trends.

1. Five viruses have now been identified that each cause a different type of hepatitis, A through E. 2. The majority of people with viral hepatitis recover uneventfully; however, in some persons with HBV and HCV, a chronic carrier state develops that may persist for a lifetime. 3. Operating room nurses are at greatest risk for HBV if they are not immunized; they can prevent HBV by obtaining hepatitis B vaccine and adhering to standard OR practices plus using eye protection.