Evidence for a role of metformin in preventing olfactory dysfunction among older adults.

BACKGROUND: Olfactory dysfunction (OD) is increasingly recognized as a hallmark of unhealthy aging and is intimately associated with mortality, but therapies remain elusive. Recognizing the increased prevalence of OD in individuals with diabetes, and the potential anti-aging effects of metformin, we studied the association of metformin use with OD. METHODS: Cross-temporal study of participants from Waves 2 (2010-11) and 3 (2015-16) of the National Social Life, Health, and Aging Project (NSHAP), a nationally representative cohort study of community-dwelling older adults. We included participants with diabetes who had complete data on olfaction and relevant covariates at Wave 2 and were not lost to follow-up at Wave 3. Olfactory identification (OI), the ability to identify the odorant, and olfactory sensitivity (OS), the ability to detect the presence of an odorant, were tested. Weighted multivariable logistic regression was used to study the association between metformin use at Wave 2 (baseline) and odds of having impaired OI/OS at Wave 3, adjusted for age, sex, race/ethnicity, education, smoking, BMI, HbA1c, years since diabetes diagnosis, and insulin use. RESULTS: Among 228 participants with diabetes (mean age=70 years, 53% female, 21% Black), 112 (49%) used metformin at baseline. Relative to nonusers, users had 58% lower odds of impaired OI and 67% lower odds of impaired OS at Wave 3. Among participants with normal baseline OS (N=62), users had 97% lower odds of impaired OS at Wave 3. CONCLUSIONS: Metformin use is associated with lower odds of OD among individuals with diabetes, suggesting a potential protective effect on olfaction. Future work including a larger sample and additional information on metformin use is needed to establish whether these findings are independent of diabetic control.

A proposed cleaning classification system for reusable medical devices to complement the Spaulding classification.

A central tenet in infection prevention is application of the Spaulding classification system for the safe use of medical devices. Initially defined in the 1950s, this system defines devices and surfaces as being critical, semi-critical or non-critical depending on how they will be used on a patient. Different levels of antimicrobial treatment, defined as various levels of disinfection or sterilization, are deemed appropriate to reduce patient risk of infection. However, a focus on microbial inactivation is insufficient to address this concern, which has been particularly highlighted in routine healthcare facility practices, emphasizing the underappreciated importance of cleaning and achieving acceptable levels of cleanliness. A deeper understanding of microbiology has evolved since the 1950s, which has led to re-evaluation of the Spaulding classification along with a commensurate emphasis on achieving appropriate cleaning. Albeit underappreciated, cleaning has always been important as the presence of residual materials on surfaces can interfere with the efficacy of the antimicrobial process to inactivate micro-organisms, as well as other risks to patients including device damage, malfunction and biocompatibility concerns. Unfortunately, this continues to be relevant, as attested by reports in the literature on the occurrence of device-related infections and outbreaks due to failures in processing expectations. This reflects, in part, increasing sophistication in device features and reuse, along with commensurate manufacturer's instructions for use. Consequently, this constitutes the first description and recommendation of a new cleaning classification system to complement use of the traditional Spaulding definitions to help address these modern-day technical and patient risk challenges. This quantitative risk-based classification system highlights the challenge of efficient cleaning based on the complexity of device features present, as an isolated variable impacting cleaning. This cleaning classification can be used in combination with the Spaulding classification to improve communication of cleaning risk of a reusable medical device between manufacturers and healthcare facilities, and improve established cleaning practices. This new cleaning classification system will also inform future creation, design thinking and commensurate innovations for the sustainable safe reuse of important medical devices.

Use of real-time immersive digital training and educational technologies to improve patient safety during the processing of reusable medical devices: Quo Vadis?

Hospital acquired infections stemming from contaminated reusable medical devices are of increasing concern. This issue is exaggerated with the introduction of complex medical devices like endoscopes and robotic instrumentation. Although medical device manufacturers validate their cleaning instructions for use, evidence in the literature demonstrates that effective device processing is not being performed consistently within sterile processing departments in clinical settings. The result is increased risks to patient safety. As a solution to this problem, focused one-on-one training increases compliance to the medical device manufacturer's processing instruction. However, often this is not a practical solution for the volume of healthcare staff responsible for device processing activities. This constitutes the first paper to address the blended use of educational and digital technologies to address these challenges and as a result inform safety and sustainability for the medical device sector. Cognitive learning theory is an evidence-based framework for learning. It supports the use of immersive educational experiences using emerging extended reality technologies (e.g., virtual or augmented reality) to increase learning comprehension. The delivery of educational content via these technologies provides an innovative option for repeatable leaning and training outcomes. The motivation is to decrease patient risk of contaminated reusable medical devices. The proposed approach while primary motivated by safety can also enhance sustainability and efficiency enabled by artificial intelligence and robotic instrumentation.

Studies on the novel effects of electron beam treated pollen on colony reproductive output in commercially-reared bumblebees (Bombus terrestris) for mass pollination applications.

Commercially-reared bumblebees provide an important pollinator service that helps support food production and security. The deployment of an appropriate non-thermal disinfection technology for the bulk treatment of pollen collected from honeybees for the feeding of commercial bumblebees is important in order to mitigate against complex diseases and unwanted pathogen spillover to native bees. High level disinfection of pollen was achieved using an electron (e)-beam dose of 100 kGy that corresponded to 78 % loss of cellular viability of bee pathogens before feeding to bumblebees as measured by the novel in vitro use of flow cytometry (FCM). Novel findings showed that e-beam treated-pollen that was fed to bumblebees produced fewer females, gynes and exhibited an absence of males when compared to control bumblebee colonies that were fed untreated commercial pollen. A similar trend emerged in bumblebee colony reproductive outputs when using membrane filtered washed pollen. Proteomic analysis of bumblebees from individual colonies fed with treated-pollen revealed a differential abundance of proteins associated with stress, immunity and metabolism when compared to the untreated pollen control group. Microbiome analysis of the bumblebee gut content revealed differences in microbiota between treated and untreated pollen in bumblebee colony studies. This novel study evaluated the impact of industrial e-beam treated-pollen on complex bee disease mitigation where physically treated-pollen fed to bumblebees was shown to substantially affect colony reproductive outputs.

A review of Spaulding's classification system for effective cleaning, disinfection and sterilization of reusable medical devices: Viewed through a modern-day lens that will inform and enable future sustainability.

Despite advances in medicine and innovations in many underpinning fields including disease prevention and control, the Spaulding classification system, originally proposed in 1957, remains widely used for defining the disinfection and sterilization of contaminated re-usable medical devices and surgical instruments. Screening PubMed and Scopus databases using a PRISMA guiding framework generated 272 relevant publications that were used in this review. Findings revealed that there is a need to evolve how medical devices are designed, and processed by cleaning, disinfection (and/or sterilization) to mitigate patient risks, including acquiring an infection. This Spaulding Classification remains in use as it is logical, easily applied and understood by users (microbiologists, epidemiologists, manufacturers, industry) and by regulators. However, substantial changes have occurred over the past 65 years that challenge interpretation and application of this system that includes inter alia emergence of new pathogens (viruses, mycobacteria, protozoa, fungi), a greater understanding of innate and adaptive microbial tolerance to disinfection, toxicity risks, increased number of vulnerable patients and associated patient procedures, and greater complexity in design and use of medical devices. Common cited examples include endoscopes that enable non- or minimal invasive procedures but are highly sophisticated with various types of materials (polymers, electronic components etc), long narrow channels, right angle and heat-sensitive components and various accessories (e.g., values) that can be contaminated with high levels of microbial bioburden and patient tissues after use. Contaminated flexible duodenoscopes have been a source of several significant infection outbreaks, where at least 9 reported cases were caused by multidrug resistant organisms [MDROs] with no obvious breach in processing detected. Despite this, there is evidence of the lack of attention to cleaning and maintenance of these devices and associated equipment. Over the last few decades there is increasing genomic evidence of innate and adaptive resistance to chemical disinfectant methods along with adaptive tolerance to environmental stresses. To reduce these risks, it has been proposed to elevate classification of higher-risk flexible endoscopes (such as duodenoscopes) from semi-critical [contact with mucous membrane and intact skin] to critical use [contact with sterile tissue and blood] that entails a transition to using low-temperature sterilization modalities instead of routinely using high-level disinfection; thus, increasing the margin of safety for endoscope processing. This timely review addresses important issues surrounding use of the Spaulding classification system to meet modern-day needs. It specifically addresses the need for automated, robust cleaning and drying methods combined with using real-time monitoring of device processing. There is a need to understand entire end-to-end processing of devices instead of adopting silo approaches that in the future will be informed by artificial intelligence and deep-learning/machine learning. For example, combinational solutions that address the formation of complex biofilms that harbour pathogenic and opportunistic microorganisms on the surfaces of processed devices. Emerging trends are addressed including future sustainability for the medical devices sector that can be enabled via a new Quintuple Helix Hub approach that combines academia, industry, healthcare, regulators, and society to unlock real world solutions.

Effects of climate and environmental variance on the performance of a novel peatland-based integrated multi-trophic aquaculture (IMTA) system: Implications and opportunities for advancing research and disruptive innovation post COVID-19 era.

Advancing wet peatland 'paludiculture' innovation present enormous potential to sustain carbon-cycles, reduce greenhouse-gas (GHG) gas emissions and to transition communities to low-carbon economies; however, there is limited scientific-evidence to support and enable direct commercial viability of eco-friendly products and services. This timely study reports on a novel, paludiculture-based, integrated-multi-trophic-aquaculture (IMTA) system for sustainable food production in the Irish midlands. This freshwater IMTA process relies on a naturally occurring ecosystem of microalgae, bacteria and duckweed in ponds for managing waste and water quality that is powered by wind turbines; however, as it is recirculating, it does not rely upon end-of-pipe solutions and does not discharge effluent to receiving waters. This constitutes the first report on the effects of extreme weather events on the performance of this IMTA system that produces European perch (Perca fluviatilis), rainbow trout (Oncorhynchus mykiis) during Spring 2020. Sampling coincided with lockdown periods of worker mobility restriction due to COVID-19 pandemic. Observations revealed that the frequency and intensity of storms generated high levels of rainfall that disrupted the algal and bacterial ecosystem in the IMTA leading to the emergence and predominance of toxic cyanobacteria that caused fish mortality. There is a pressing need for international agreement on standardized set of environmental indicators to advance paludiculture innovation that addresses climate-change and sustainability. This study describes important technical parameters for advancing freshwater aquaculture (IMTA), which can be future refined using real-time monitoring-tools at farm level to inform management decision-making based on evaluating environmental indicators and weather data. The relevance of these findings to informing global sustaining and disruptive research and innovation in paludiculture is presented, along with alignment with UN Sustainable Development goals. This study also addresses global challenges and opportunities highlighting a commensurate need for international agreement on resilient indicators encompassing linked ecological, societal, cultural, economic and cultural domains.

Nonpharmacological interventions to reduce respiratory viral transmission: an evidence-based review with recommendations.

BACKGROUND: Viral respiratory infections are a leading cause of worldwide mortality and exert the potential to cause global socioeconomic crises. However, inexpensive, efficacious, and rapidly deployable strategies to reduce viral transmission are increasingly important in the setting of an ongoing pandemic, though not entirely understood. This article provides a comprehensive review of commonly employed nonpharmacological interventions to interrupt viral spread and provides evidence-based recommendations for their use. METHODOLOGY: A systematic review of three databases was performed. Studies with defined endpoints of subjects receiving one of five interventions (nasal washing, gargling, personal protective equipment (PPE), social distancing, and hand hygiene) were included. An evidence-based review of the highest level of evidence, with recommendations, was created in accordance with a previously described, rigorous, iterative process. RESULTS: Fifty-four primary studies were included. The most commonly studied intervention was hand hygiene, followed by PPE, gargling, saline nasal washing, and social distancing. CONCLUSIONS: Mask use and hand hygiene are strong recommendations for prevention of viral transmission. Donning gloves, gowns, and eye protection are a recommendation in healthcare settings. Saline nasal washing and gargling are options in selected populations. Although an aggregate level of evidence is not provided, the authors recommend social distancing.

Opportunities for the application of real-time bacterial cell analysis using flow cytometry for the advancement of sterilization microbiology.

Medical devices provide critical care and diagnostic applications through patient contact. Sterility assurance level (SAL) may be defined as the probability of a single viable micro-organism occurring on an item after a sterilization process. Sterilization microbiology often relies upon using an overkill validation method where a 12-log reduction in recalcitrant bacterial endospore population occurs during the process that exploits conventional laboratory-based culture media for enumeration. This timely review explores key assumptions underpinning use of conventional culture-based methods in sterilization microbiology. Consideration is given to how such methods may limit the ability to fully appreciate the inactivation kinetics of a sterilization process such as vaporized hydrogen peroxide (VH2O2) sterilization, and consequently design efficient sterilization processes. Specific use of the real-time flow cytometry (FCM) is described by way of elucidating the practical relevance of these limitation factors with implications and opportunities for the sterilization industry discussed. Application of FCM to address these culture-based limitation factors will inform real-time kinetic inactivation modelling and unlock potential to embrace emerging opportunities for pharma, medical device and sterilization industries including potentially disruptive applications that may involve reduced usage of sterilant.

Purified ß-glucans from the Shiitake mushroom ameliorates antibiotic-resistant Klebsiella pneumoniae-induced pulmonary sepsis.

Bacterial infection remains the main cause of acute respiratory distress syndrome and is a leading cause of death and disability in critically ill patients. Here we report on the use of purified ß-glucan (lentinan) extracts from Lentinus edodes (Shiitake) mushroom that can reduce infection by a multidrug-resistant clinical isolate of Klebsiella pneumoniae in a rodent pneumonia model, likely through immunomodulation. Adult male Sprague-Dawley rats were subjected to intra-tracheal administration of K. pneumoniae to induce pulmonary sepsis and randomized to three groups; vehicle control (Vehicle, n = 12), commercial lentinan (CL, n = 8) or in-house extracted lentinan (IHL, n = 8) were administered intravenously 1 h postinfection. Physiological parameters and blood gas analysis were measured, bacterial counts from bronchoalveolar-lavage (BAL) were determined, along with differential staining of white cells and measurement of protein concentration in BAL 48 h after pneumonia induction. Use of IHL extract significantly decreased BAL CFU counts. Both CL and IHL extractions reduced protein concentration in BAL. Use of IHL resulted in an improvement in physiological parameters compared to controls and CL. In conclusion, administration of lentinan to treat sepsis-induced lung injury appears safe and effective and may exert its effects in an immunomodulatory manner.

Lack of impact of radiologic septal measurements upon patient symptoms and performance of septoplasty during endoscopic sinus surgery.

BACKGROUND: Recent literature suggests that concurrent septoplasty during endoscopic sinus surgery (ESS) improves patient outcomes, however, the underlying indications for performing concurrent septoplasty are unknown. The objective of this study was to investigate the relationship between objective radiologic measures of nasal septal deviation with preoperative patient symptomatology and measures of CRS disease severity. We also sought to understand the association of objective radiologic measurements with surgeon performance of concurrent septoplasty during ESS. METHODOLOGY: Seventy-four patients with CRS undergoing ESS were prospectively enrolled. Angles of septal deviation, intranasal areas and volumes were assessed on preoperative computed tomography (CT) scans and correlated with a robust battery of patient reported outcomes measures (PROMs), objective measures of CRS severity including olfaction scores, radiologic and endoscopic staging, and performance of septoplasty. RESULTS: Intranasal areas and volumes demonstrated only weak linear associations with patient-reported nasal congestion, however, angles of septal deviation alone did not correlate with congestion or any other PROM measure. Meanwhile, radiologic septal-related measurements did not correlate with objective measures of CRS disease severity or the performance of a concurrent septoplasty. CONCLUSIONS: Though prior studies demonstrate improved patient outcomes in the setting of concurrent septoplasty during ESS, this study failed to establish an association between preoperative radiologic septal-related measurements and patient symptomatology or surgeon decision to perform septoplasty. Although objective factors to identify patients most likely to benefit from concurrent septoplasty remain unidentified, the potential improvement of surgical recommendations and patient outcomes makes this an important area of continued investigation.

Terminal sterilization of medical devices using vaporized hydrogen peroxide: a review of current methods and emerging opportunities.

Medical devices are an important and growing aspect of healthcare provision and are increasing in complexity to meet established and emerging patient needs. Terminal sterilization plays a vital role in the provision of safe medical devices. While terminal sterilization technologies for medical devices include multiple radiation options, ethylene oxide remains the predominant nonthermal gaseous option, sterilizing c. 50% of all manufactured devices. Vaporized hydrogen peroxide (abbreviated VH2O2 by the International Organization for Standardization) is currently deployed for clinical sterilization applications, where its performance characteristics appear aligned to requirements, constituting a viable alternative low-temperature process for terminal processing of medical devices. However, VH2O2 has operational limitations that create technical challenges for industrial-scale adoption. This timely review provides a succinct overview of VH2O2 in gaseous sterilization and addresses its applicability for terminal sterilization of medical devices. It also describes underappreciated factors such as the occurrence of nonlinear microbial inactivation kinetic plots that may dictate a need to develop a new standard approach to validate VH2O2 for terminal sterilization of medical devices.

Silver nanoparticles in the environment: Sources, detection and ecotoxicology.

The environmental impact of silver nanoparticles (AgNP) has become a topic of interest recently, this is due to the fact that AgNPs have been included in numerous consumer products including textiles, medical products, domestic appliances, food containers, cosmetics, paints and nano-functionalised plastics. The production, use and disposal of these AgNP containing products are potential routes for environmental exposure. These concerns have led to a number of studies investigating the release of particles from nano-functionalised products, the detection of the particles in the aquatic environment and the potential environmental toxicology of these AgNPs to aquatic organisms. The overall aim of this review is to examine methods for the capture and detection of AgNPs, potential toxicity and transmission routes in the aquatic environment.

Detection, fate and inactivation of pathogenic norovirus employing settlement and UV treatment in wastewater treatment facilities.

It is accepted that discharged wastewaters can be a significant source of pathogenic viruses in receiving water bodies contributing to pollution and may in turn enter the human food chain and pose a risk to human health, thus norovirus (NoV) is often a predominant cause of gastroenteritis globally. Working with NoV poses particular challenges as it cannot be readily identified and detection by molecular methods does not assess infectivity. It has been proposed that the infectivity of NoV may be modelled through the use of an alternative virus; F-specific RNA (FRNA) bacteriophages; GA genotype and other FRNA bacteriophages have been used as a surrogate in studies of NoV inactivation. This study investigated the efficiency of novel pulsed ultraviolet irradiation and low pressure ultraviolet irradiation as a potential pathogen inactivation system for NoV and FRNA bacteriophage (GA) in secondary treated wastewaters. The role of UV dose and the impact of suspended solids concentration on removal efficiency were also examined. The study also investigated the role of settlement processes in wastewater treatment plants in removing NoV. While NoV inactivation could not be determined it was found that at a maximum UV dose of 6.9J/cm(2) (6900mJ/cm(2)) an average 2.4 log removal of FRNA bacteriophage (GA) was observed; indicating the potential need for high UV doses to remove NoV if FRNA bacteriophage prove a suitable indicator for NoV. The study found that increasing concentrations of suspended solids impacted on PUV efficiency however, it appears the extent of the impact may be site specific. Furthermore, the study found that settlement processes can play a significant role in the removal of FRNA bacteriophage, thus potentially NoV.

The pulsed light inactivation of veterinary relevant microbial biofilms and the use of a RTPCR assay to detect parasite species within biofilm structures.

The presence of pathogenic organisms namely parasite species and bacteria in biofilms in veterinary settings, is a public health concern in relation to human and animal exposure. Veterinary clinics represent a significant risk factor for the transfer of pathogens from housed animals to humans, especially in cases of wound infection and the shedding of faecal matter. This study aims to provide a means of detecting veterinary relevant parasite species in bacterial biofilms, and to provide a means of disinfecting these biofilms. A real time PCR assay was utilized to detect parasite DNA in Bacillus cereus biofilms on stainless steel and PVC surfaces. Results show that both Cryptosporidium and Giardia attach to biofilms in large numbers (100-1000 oo/cysts) in as little as 72 hours. Pulsed light successfully inactivated all test species (Listeria, Salmonella, Bacillus, Escherichia) in planktonic and biofilm form with an increase in inactivation for every increase in UV dose.

Investigation of critical inter-related factors affecting the efficacy of pulsed light for inactivating clinically relevant bacterial pathogens.

AIMS: To investigate critical electrical and biological factors governing the efficacy of pulsed light (PL) for the in vitro inactivation of bacteria isolated from the clinical environment. Development of this alternative PL decontamination approach is timely, as the incidence of health care-related infections remains unacceptably high. METHODS AND RESULTS: Predetermined cell numbers of clinically relevant Gram-positive and Gram-negative bacteria were inoculated separately on agar plates and were flashed with

Organocatalytic enantioselective total synthesis of (-)-arboricine.

The tetracyclic indole alkaloid (-)-arboricine has been prepared using an asymmetric organocatalytic Pictet-Spengler reaction as the key step followed by a diastereoselective Pd-catalyzed iodoalkene/enolate cyclization. The absolute stereochemistry was unequivocally proven by X-ray crystallographic analysis and appeared to be opposite to the published structure in the original paper.

Evidence of lethal and sublethal injury in food-borne bacterial pathogens exposed to high-intensity pulsed-plasma gas discharges.

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to pulsed-plasma gas discharges (PPGD). METHODS AND RESULTS: The fluorescent redox probe 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4 degrees C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct-selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log-unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high-intensity PPGD treatments.

Pulsed-plasma gas-discharge inactivation of microbial pathogens in chilled poultry wash water.

A pulsed-plasma gas-discharge (PPGD) system was developed for the novel decontamination of chilled poultry wash water. Treatment of poultry wash water in the plasma generation chamber for up to 24 s at 4 degrees C reduced Escherichia coli NCTC 9001, Campylobacter jejuni ATCC 33560, Campylobacter coli ATCC 33559, Listeria monocytogenes NCTC 9863, Salmonella enterica serovar Enteritidis ATCC 4931, and S. enterica serovar Typhimurium ATCC 14028 populations to non-detectable levels (< or = 8 log CFU/ml). Although similar PPGD treatments at 4 degrees C also produced significant reductions (> or = 3 log CFU/ml) in recalcitrant B. cereus NCTC 11145 endospore numbers within 30 s, the level of endospore reduction was dependent on the nature of the sparged gas used in the plasma treatments. Scanning electron microscopy revealed that significant damage occurred at the cellular level in PPGD-treated test organisms. This electrotechnology delivers energy in intense ultrashort bursts, generating products such as ozone, UV light, acoustic and shock waves, and pulsed electric fields that have multiple bactericidal properties. This technology offers an exciting complementary or alternative approach for treating raw poultry wash water and for preventing cross-contamination in processing environments.

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields.

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae.

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.