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Topical antimicrobials that reduce the bacterial bioburden within a chronically-infected wound may have helpful or harmful effects on the healing process. We used murine models of full-thickness skin wounds to determine the effects of the novel biofilm-dispersing wound gel (BDWG) and its gel base on the healing of uninfected wounds. The rate of wound closure over 19 days was comparable among the BDWG-treated (BT) wounds and the controls. Compared with the controls, histology of the BT wounds showed formation of a stable blood clot at day 1, more neovascularisation and reepithelialisation at day 3, and more organised healing at day 7. Fluorescence-activated cell sorting analysis showed a lower percentage of neutrophils in wounded tissues of the BT group at days 1 and 3, and significantly more M2 macrophages at day 3. Levels of proinflammatory cytokines and chemokines were increased over the uninjured baseline within the wounds of all treatment groups but the levels were significantly lower in the BT group at day 1, modulating the inflammatory response. Our results suggest that BDWG does not interfere with the wound healing process and may enhance it by lowering inflammation and allowing transition to the proliferative stage of wound healing by day 3.

The gold standard for microbial detection in prosthetic joint infections is the multiple culture of the peri-prosthetic tissue. The fluid cultures after sonication can improve the recovery of the microorganisms. The aim of this study was to evaluate the sonication technique with a plastic bag and the effect of refrigeration on microorganism detection with conventional culturing, MALDI-TOF MS and qPCR assay on an orthopedic screw model. We produced biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans on orthopedic screws, which were stored under different conditions and temperatures before sonication. After sonication, the mass spectrometry by MALDI-TOF, qPCR and culture protocols was performed using the sonicated fluid, for detecting the microorganisms involved in the biofilm. The bacterial bioburden decreased by approximately one log after the refrigeration period, in the screws containing P. aeruginosa and S. aureus biofilms. All the microorganisms involved in the screw biofilms were detected with MALDI-TOF and qPCR. Significant reductions in CFU counts occurred only in groups stored in the plastic bag, indicating that changes in temperature and humidity may favor cell death. However, this variation is not important for this model as it did not affect the detection owing to the high counts obtained. Microbial identification by MALDI-TOF in sonicated fluid is feasible. With qPCR, there were no differences between the detection in the screws processed immediately or after refrigeration. It is necessary to consider whether or not the refrigeration period would affect microbial recovery in an explanted prosthesis.

In recent years there has been increasing advocacy for highly immunogenic gamma-irradiated vaccines, several of which are currently in clinical or pre-clinical trials. Importantly, various methods of mathematical modelling and sterility testing are employed to ensure sterility. However, these methods are designed for materials with a low bioburden, such as food and pharmaceuticals. Consequently, current methods may not be reliable or applicable to estimate the irradiation dose required to sterilize microbiological preparations for vaccine purposes, where bioburden is deliberately high. In this study we investigated the applicability of current methods to calculate the sterilizing doses for different microbes. We generated inactivation curves that demonstrate single-hit and multiple-hit kinetics under different irradiation temperatures for high-titre preparations of pathogens with different genomic structures. Our data demonstrate that inactivation of viruses such as Influenza A virus, Zika virus, Semliki Forest virus and Newcastle Disease virus show single-hit kinetics following exposure to gamma-irradiation. In contrast, rotavirus inactivation shows multiple-hit kinetics and the sterilizing dose could not be calculated using current mathematical methods. Similarly, Streptococcus pneumoniae demonstrates multiple-hit kinetics. These variations in killing curves reveal an important gap in current mathematical formulae to determine sterility assurance levels. Here we propose a simple method to calculate the irradiation dose required for a single log10 reduction in bioburden (D10) value and sterilizing doses, incorporating both single- and multiple-hit kinetics, and taking into account the possible existence of a resistance shoulder for some pathogens following exposure to gamma-irradiation.

This review of the literature concerning bacteria, antibiotics and tissue repair shows there are extensive data supporting microbial interference with wound healing once bacterial burden exceeds 104 CFU per unit of measure, The mechanism of bacterial interference lies largely in prolonging the inflammatory phase of tissue repair. Reducing the microbial bioburden allows tissue repair to continue. Systemic and topical antimicrobials appear critical to reducing the bioburden and facilitating repair. The current controversy over the use of antimicrobials in patients with chronically infected wounds, in particular, revolves around the definition of infection. The reliance on classic clinical signs of inflammation to support antimicrobial use in these patients is tenuous due to the lack of correlation of these signs with the microbial burden known to impair tissue repair.

Summary Background Pathogens involved in healthcare-associated infections can quickly spread in the environment, particularly to frequently touched surfaces, which can be reservoirs for pathogens. Aim The purpose of this study was to investigate naturally occurring bacterial contamination on touch surfaces in five French long-term care facilities and to compare bacterial populations recovered from copper and control surfaces. Methods More than 1300 surfaces were sampled. The collected bacteria were identified to obtain a global view of the cultivable bacterial populations colonizing touch surfaces. Haemolytic colonies and putative pathogens were also screened using specific agar plates and then identified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In total, more than 3400 colonies were analysed. Findings Staphylococcus and Micrococcus were the two predominant genera present on touch surfaces, respectively occurring on 51.8% and 48.0% of control surfaces. In these facilities with relatively low bioburden, copper surfaces efficiently reduced the occurrence frequencies of three genera: Staphylococcus, Streptococcus and Roseomonas. Pathogenic species such as Staphylococcus aureus, Enterococcus faecalis and E. faecium were observed in very few samples. In addition, meticillin-resistant S. aureus was observed on five control surfaces and one copper surface. Conclusion Contamination of healthcare facilities touch surfaces can be the source for the spread of bacteria through the institution. This in situ study shows that the frequency of the contamination as well as the specific bacterial population bioburden is reduced on copper alloy surfaces.

In pharmaceutical industry antimicrobial preservatives that restrict multiplication of microorganisms and decrease bioburden are usually added in composition of hydrophilic medicines that include components of natural origin to prevent possible risks of functional changes in drugs characteristics caused by microbial contamination. Therefore, microbiological purity (MBP) is a crucial criteria for medicines quality. The purpose of the work was to develop the MBP testing methodology for complex gel containing dense extract of hypericum herbs and calendula flowers using structural-mechanical and technological studies. Methodology for MBP testing of proposed gel was developed and justified considering criteria given in State Pharmacopoeia of Ukraine 2.0. Originality of MPT methodology consists in the gel’s antimicrobial action neutralization to decrease possible risks of false results occurrence (including microorganisms inhibited by antimicrobial action of the gel). The applicability of the methodology was determined comparing the number of test cultures CFUs in presence of gel under specified condition with number of CFUs in positive control. The methodology is suitable for microbiological purity testing of the sample, as well as for the Preservatives Efficacy Testing (PET). When applying the developed methodology, the antimicrobial action of the sample is completely eliminated. While developing the MPT methodology compliance with following acceptance criteria was confirmed for tested sample: Absence of Staphylococcus aureus (in 1 g); Absence of Pseudomonas aeruginosa (in 1 g); Total aerobic microbial count (TAMC) – 10² CFU per 1 g; Total combined yeasts and molds count (TYMC) – 10¹ CFU per 1 g. All the tested samples met the requirements of the 2.0. version of State Pharmacopoeia of Ukraine for non-sterile dosage forms for oromucosal and dermal use.

Online water bioburden analyzers (OWBAs) can provide real-time feedback on viable bacteria in high-purity water (HPW) systems for pharmaceutical manufacturers. To calibrate and validate OWBAs, which detect bacteria using scattered light and bacterial autofluorescence, standards are needed that mimic the characteristics of bacteria in HPW. To guide selection of potential standards, e.g., fluorescent microspheres, a relevant bacterial contaminant, Ralstonia pickettii, was characterized for size, count, viability, and autofluorescence after exposure for 24 h to HPW or a nutrient environment. The cells exposed to HPW showed smaller sizes, with lower counts and autofluorescence intensities, but similar spectral features. The cell characteristics are discussed in comparison with a set of fluorescent microspheres, considering factors relevant to OWBAs. These studies suggest that fluorescent microspheres should be relatively small (

Hemoglobin microparticles (HbMP) produced with a three-step procedure including co-precipitation of hemoglobin with manganese carbonate, protein crosslinking and dissolution of the carbonate template were shown to be suitable for application as artificial oxygen carriers. First preclinical safety investigations delivered promising results. Bacterial safety plays a decisive role during the production of the HbMP. Therefore, bioburden and endotoxin content of the starting materials (especially hemoglobin) and the final particle suspension are intensively tested. However, some bacteria may not have been detected by the standard tests due to low concentration. The aim of this study was to investigate how these bacteria would behave in the fabrication process. Biocidal effects are known for glutaraldehyde and for ethylenediaminetetraacetic acid, chemicals that are used in the fabrication process of HbMP. It could be shown that both chemicals prevent bacterial growth at the concentrations used during the HbMP fabrication. In addition, the particle production was carried out with hemoglobin solutions spiked with Escherichia coli or Staphylococcus epidermidis. No living bacteria could be detected in the final particle suspensions. Therefore, we conclude that the HbMP fabrication procedure is safe in respect of a bacterial contamination.

Objective:To investigate the frequency of environmental contamination in hospital areas outside patient rooms and in outpatient healthcare facilities.Design:Culture survey.Setting:This study was conducted across 4 hospitals, 4 outpatient clinics, and 1 surgery center.Methods:We conducted 3 point-prevalence culture surveys for methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Clostridioides difficile, Candida spp, and gram-negative bacilli including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Stenotrophomonas maltophilia in each facility. In hospitals, high-touch surfaces were sampled from radiology, physical therapy, and mobile equipment and in emergency departments, waiting rooms, clinics, and endoscopy facilities. In outpatient facilities, surfaces were sampled in exam rooms including patient and provider areas, patient bathrooms, and waiting rooms and from portable equipment. Fluorescent markers were placed on high-touch surfaces and removal was assessed 1 day later.Results:In the hospitals, 110 (9.4%) of 1,195 sites were positive for 1 or more bacterial pathogens (range, 5.3%–13.7% for the 4 hospitals) and 70 (5.9%) were positive for Candida spp (range, 3.7%–5.9%). In outpatient facilities, 31 of 485 (6.4%) sites were positive for 1 or more bacterial pathogens (range, 2% to 14.4% for the 5 outpatient facilities) and 50 (10.3%) were positive for Candida spp (range, 3.9%–23.3%). Fluorescent markers had been removed from 33% of sites in hospitals (range, 28.4%–39.7%) and 46.3% of sites in outpatient clinics (range, 7.4%–82.8%).Conclusions:Surfaces in hospitals outside patient rooms and in outpatient facilities are frequently contaminated with healthcare-associated pathogens. Improvements in cleaning and disinfection practices are needed to reduce contamination.

The aim of this study was to determine the epidemiology (location, microbial load, microbiome, presence/absence of biofilm and pathogens, including ESKAPE-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species, and antimicrobial susceptibility profiles) of the bacterial contamination on intensive care units (ICUs) surfaces. Fifty-seven high-touched surfaces were collected from adult, paediatric and neonatal ICUs from two large public Brazilian hospitals from central and north regions. Samples (c. 4 cm2 ) were subjected to culture (qualitative), qPCR targeting 16s rRNA gene (microbial load-bacteria per cm2 ), 16s rRNA amplicon sequencing (microbiome analysis) and scanning electron (SEM) or confocal laser scanning microscopy (CLSM) (biofilm presence). Multidrug resistant organisms (MROs) were detected using specific chromogenic agar. The average bacterial load was 1·32 × 104 bacteria per cm2 , container for newborn feeding bottles, stretcher mattress, humidicrib mattress filling and computer keyboards presented the higher bioburden. However, only 45·6% (26/57) were culture-positive, including 4/26 with MROs. ESKAPE organisms were detected in 51·8% of the samples subjected to next-generation sequencing. Viability staining and CLSM demonstrated live bacteria on 76·7% of culture-negative samples. Biofilm was present on all surfaces subjected to microscopy (n = 56), demonstrating that current cleaning practices are suboptimal and reinforcing that MROs are incorporated into hospital surfaces biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of healthcare facilities surfaces has been shown to play a major role in transmission of pathogens. The findings of this study show that dry surface biofilms are widespread and can incorporate pathogens and multidrug-resistant organisms (MROs). Biofilms on highly touched surfaces pose a risk to patients, as dry surface biofilms persist for long period and micro-organisms within biofilm have been shown to be transmitted. This study also provides a better understanding of microbial populations in hospital environments, reinforcing that pathogens and MROs are found incorporated into biofilm, which impacts the difficulty in cleaning/disinfection.

Background Environmental disinfection with continuously antimicrobial surfaces could offer superior control of surface bioburden. We sought to decide the efficacy of photocatalyst antimicrobial coating in reducing methicillin-resistant Staphylococcus aureus (MRSA) acquisition in high incidence setting. Methods We performed prospective cohort study involving patients hospitalized in medical intensive care unit. A titanium dioxide-based photocatalyst was coated on high touch surfaces and walls. Five months of pre-intervention data were compared with five months of post-intervention data. The incidence rates of multidrug-resistant organism acquisition and the rates of hospital-acquired blood stream infection, pneumonia, urinary tract infection, and Clostridium difficile–associated diseases were compared using Cox proportional hazards regression analysis. Results In total, 621 patients were included. There was significant decrease in MRSA acquisition rate after photocatalyst coating (hazard ratio, 0.37; 95% confidence interval, 0.14–0.99; p = 0.04). However, clinical identification of vancomycin-resistant Enterococcus spp. and multidrug-resistant Acinetobacter baumannii did not decrease significantly. The hazard of contracting hospital-acquired pneumonia during the intervention period compared to baseline period was 0.46 (95% confidence interval, 0.23–0.94; p = 0.03). Conclusions In conclusion, MRSA rate was significantly reduced after photocatalyst coating. We provide evidence that photocatalyst disinfection can be an adjunctive measure to control MRSA acquisition in high-incidence settings. Trial registration ISRCTN Registry (ISRCTN31972004). Registered retrospectively on November 19, 2018. Electronic supplementary material The online version of this article (10.1186/s12879-018-3555-1) contains supplementary material, which is available to authorized users.

The study was aimed to evaluate the effectiveness of maggot therapy in healing of cutaneous infected wound in streptozotocin (STZ) induced diabetic Wistar rat. For live maggots, the sterilized eggs of Lucilia sericata were obtained from colonies established in laboratory. Diabetes model was established in 48 male Wister rat by intra-peritoneal injection of STZ at the dose of 60 mg/kg body-weight. Cutaneous wounds exposed with mixed colonies of bacteria like Staphylococcus aureus, E. coli and Pseudomonas aeruginosa were prepared in all rat. The animals equally divided in 4 groups with 12 rats each being presented as treatment group of control, antibiotic, maggot and maggot with antibiotic in combination. All treatments were done once and hold for 24 hours. Wound kinetics and bacterial bio burden were measured at weekly interval to till complete healing. Significant reduction in wound area with maximum contraction was found (>95%) in maggot treated group when compared to antibiotic treated (79%) and control (72%). In maggot as well as maggot and antibiotic in combination group showed early elimination of bacterial bio-burden 7.88±0.03log CFU/ml to 1.12±0.65log CFU/ml and 7.86±0.04) log CFU/ml to 1.54±0.52log CFU/ml respectively in three weeks of time. Early healing indication was also experienced on histomorphological examination of wounded tissue of maggot treated groups by early and better epithelialization, collagenation and neovascularization with complete healing of wound in three weeks in comparison to antibiotic and control respectively. However, the present study did not show any difference in healing of wound with use of maggot alone or in antibiotic combination. Live maggot of Lucilia sericata effectively lower bacterial bioburden and and accelerate healing of infected cutaneous wound in diabetic conditions.

Background The skin on human feet presents unique environments for the proliferation of potentially pathogenic commensals. This study examined microflora changes on healthy intact skin under a semiocclusive dressing on the medial longitudinal arch of the foot to determine changes in growth, distribution, and frequency of microflora under the dressing. Methods Nine human participants wore a low-adherent, absorbent, semiocclusive dressing on the medial longitudinal arch of the left foot for 2 weeks. An identical location on the right foot was swabbed and used as a control. Each foot was swabbed at baseline, week 1, and week 2. The swabs were cultured for 48 hours. Visual identification, Gram staining, DNase test agar, and a latex slide agglutination test were used to identify genera and species. Results Microflora growth was categorized as scant (0–10 colony-forming units [CFU]), light (11–50 CFU), moderate (51–100 CFU), or heavy (>100 CFU). Scant and light growth decreased and moderate and heavy growth increased under the dressing compared with the control. Seven different genera of bacteria were identified. Coagulase-negative Staphylococcus spp appeared most frequently, followed by Corynebacterium spp. Conclusions Changes in microflora distribution, frequency, and growth were found under the dressing, supporting historical studies. Microflora changes were identified as an increase in bioburden and reduction in diversity. The application of similar methods, using more sophisticated identification and analysis techniques and a variety of dressings, could lead to a better understanding of bacterial and fungal growth under dressings, informing better dressing selection to assist the healing process of wounds and prevent infection.

Sampling campaigns indoors have shown that occupants exposed to contaminated air generally exhibit diverse health outcomes. This study intends to assess the deposition rates of total settleable dust and bioburden in the indoor air of dwellings onto quartz fiber filters and electrostatic dust collectors (EDCs), respectively. EDC extracts were inoculated onto malt extract agar (MEA) and dichloran glycerol (DG18) agar-based media used for fungal contamination characterization, while tryptic soy agar (TSA) was applied for total bacteria assessment, and violet red bile agar (VRBA) for Gram-negative bacteria. Azole-resistance screening and molecular detection by qPCR was also performed. Dust loading rates ranged from 0.111 to 3.52, averaging 0.675 μg cm−2 day−1. Bacterial counts ranged from undetectable to 16.3 colony-forming units (CFU) m−2 day−1 and to 2.95 CFU m−2 day−1 in TSA and VRBA, respectively. Fungal contamination ranged from 1.97 to 35.4 CFU m−2 day−1 in MEA, and from undetectable to 48.8 CFU m−2 day−1 in DG18. Penicillium sp. presented the highest prevalence in MEA media (36.2%) and Cladosporium sp. in DG18 (39.2%). It was possible to observe: (a) settleable dust loadings and fungal contamination higher in dwellings with pets, (b) fungal species considered indicators of harmful fungal contamination, (c) Aspergillus section Candidi identified in supplemented media with voriconazole and posaconazole, (d) specific housing typologies and (e) specific housing characteristics influencing the microbial contamination.

The antibiotic resistance of an organism has become an endeavor worldwide. The resistance of bacteria is impacting the immunocompromised patient, especially with diabetic. The bacteriophages are more specific in the elimination of the infections caused by the organism. One of the significant bacteria is Vancomycin-resistant Enterococcus faecalis (VREF), emerging as bioburden rendering infectious diseases in developing countries. We attempted to treat infection of VREF in the induced and non-induced diabetic mice model along with antibiotic and phage treatment. The phage has shown more efficiency than vancomycin antibiotic alone and combined therapy with antibiotic phage treated. The considering phage therapy an alternative source for the treatment in the insulin-dependent diabetic with an infection of bacteria.

During the current COVID-19 pandemic, the use of face masks has become increasingly recommended and even mandatory in community settings. To evaluate the risk of bacterial cross-contamination, this study analyzed the bacterial bioburden of disposable surgical masks and homemade cotton masks, and surveyed the habits and face mask preferences of the Flemish population. Using culture approaches and 16S rRNA gene amplicon sequencing, we analyzed the microbial community on surgical and/or cotton face masks of 13 healthy volunteers after 4 h of wearing. Cotton and surgical masks contained on average 1.46 × 105 CFU/mask and 1.32 × 104 CFU/mask, respectively. Bacillus, Staphylococcus, and Acinetobacter spp. were mostly cultured from the masks and 43% of these isolates were resistant to ampicillin or erythromycin. Microbial profiling demonstrated a consistent difference between mask types. Cotton masks mainly contained Roseomonas, Paracoccus, and Enhydrobacter taxa and surgical masks Streptococcus and Staphylococcus. After 4 h of mask wearing, the microbiome of the anterior nares and the cheek showed a trend toward an altered beta-diversity. According to dedicated questions in the large-scale Corona survey of the University of Antwerp with almost 25,000 participants, only 21% of responders reported to clean their cotton face mask daily. Laboratory results indicated that the best mask cleaning methods were boiling at 100°C, washing at 60°C with detergent or ironing with a steam iron. Taken together, this study suggests that a considerable number of bacteria, including pathobionts and antibiotic resistant bacteria, accumulate on surgical and even more on cotton face masks after use. Based on our results, face masks should be properly disposed of or sterilized after intensive use. Clear guidelines for the general population are crucial to reduce the bacteria-related biosafety risk of face masks, and measures such as physical distancing and increased ventilation should not be neglected when promoting face mask use.

© 2020 Elsevier Ltd. All rights reserved., A wider characterization of indoor air quality during sleep is still lacking in the literature. This study intends to assess bioburden before and after sleeping periods in Portuguese dwellings through active methods (air sampling) coupled with passive methods, such as electrostatic dust cloths (EDC); and investigate associations between before and after sleeping and bioburden. In addition, and driven by the lack of information regarding fungi azole-resistance in Portuguese dwellings, a screening with supplemented media was also performed. The most prevalent genera of airborne bacteria identified in the indoor air of the bedrooms were Micrococcus (41%), Staphylococcus (15%) and Neisseria (9%). The major indoor bacterial species isolated in all ten studied bedrooms were Micrococcus luteus (30%), Staphylococcus aureus (13%) and Micrococcus varians (11%). Our results highlight that our bodies are the source of the majority of the bacteria found in the indoor air of our homes. Regarding air fungal contamination, Chrysosporium spp. presented the highest prevalence both in after the sleeping period (40.8%) and before the sleeping period (28.8%) followed by Penicillium spp. (23.47% morning; 23.6% night) and Chrysonilia spp. (12.4% morning; 20.3% night). Several Aspergillus sections were identified in air and EDC samples. However, none of the fungal species/strains (Aspergillus sections Fumigati, Flavi, Nidulantes and Circumdati) were amplified by qPCR in the analyzed EDC. The correlations observed suggest reduced susceptibility to antifungal drugs of some fungal species found in sleeping environments. Toxigenic fungal species and indicators of harmful fungal contamination were observed in sleeping environments., This work was supported by Instituto Politécnico de Lisboa, Lisbon, Portugal for funding the Project "E2SleepeThe impact of environmental exposure on sleep” (IPL/2017/E2SLEEP/ESTeSL/711030) and the Project “Occupational exposure of ambulance drivers to bioburden” (IPL/2020/BIO-AmbuDrivers_ESTeSL). H&TRC authors gratefully acknowledge the FCT/MCTES national support through the UIDB/05608/2020 and UIDP/05608/2020. N. Canha also acknowledges the national FCT funding for his Post doc grant (SFRH/BPD/102944/2014) and his contract ISD-ID (IST-ID/098/2018). The FCT support is also acknowledged by C2TN/IST (UIDB/04349/2020+UIDP/04349/2020) and CESAM (UIDB/50017/2020+UIDP/50017/2020). This study also had the support of LIFE Index-Air project (LIFE15 ENV/PT/000674). It reflects only the view of the authors and EASME is not responsible for any use that may be made of the information it contains.

Background The Spacecraft Assembly Facility (SAF) at the NASA’s Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. Results Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. Conclusion This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays.

Monitoring campaigns in several buildings have shown that occupants exposed to contaminated indoor air generally exhibit diverse health symptoms. This study intends to assess settleable dust loading rates and bioburden in Portuguese dwellings by passive sampling onto quartz fiber filters and electrostatic dust cloths (EDCs), respectively. Settled dust collected by EDCs was analyzed by culture-based methods (including azole-resistance screening) and qPCR, targeting four different toxigenic Aspergillus sections (Flavi, Fumigati, Circumdati, and Nidulantes). Dust loading rates and bioburden showed higher variability in the summer season. In both seasons, Penicillium sp. was the one with the highest prevalence (59.1% winter, 58.1% summer), followed by Aspergillus sp. in winter (13.0%). Fungal contamination increased in the winter period, while bacterial counts decreased. Aspergillus sections Circumdati and Nidulantes, detected in voriconazole supplemented media, and Aspergillus sections Fumigati and Nidulantes, detected by molecular tools, were found in the winter samples. This study reinforces the importance of applying: (a) Passive sampling methods in campaigns in dwellings, (b) two different culture media (MEA and DG18) to assess fungi, (c) in parallel, molecular tools targeting the most suitable indicators of fungal contamination, and (d) azole resistance screening to unveil azole resistance detection in fungal species.

A spacecraft is a confined system that is inhabited by a changing microbial consortium, mostly originating from life-supporting devices, equipment collected in pre-flight conditions, and crewmembers. Continuous monitoring of the spacecraft’s bioburden employing culture-based and molecular methods has shown the prevalence of various taxa, with human skin-associated microorganisms making a substantial contribution to the spacecraft microbiome. Microorganisms in spacecraft can prosper not only in planktonic growth mode but can also form more resilient biofilms that pose a higher risk to crewmembers’ health and the material integrity of the spacecraft’s equipment. Moreover, bacterial biofilms in space conditions are characterized by faster formation and acquisition of resistance to chemical and physical effects than under the same conditions on Earth, making most decontamination methods unsafe. There is currently no reported method available to combat biofilm formation in space effectively and safely. However, antibacterial photodynamic inactivation based on natural photosensitizers, which is reviewed in this work, seems to be a promising method.

The assessment and control of microbial contamination in health care facilities is presently a mandatory and vital part of strategies to prevent and control hospital-acquired infections. This study aims to assess the bioburden with two passive sampling methods (30 ventilations grids swabs and 16 electrostatic dust collectors (EDCs)) at Clinical Pathology Services. The fungal burden was characterized through molecular tools, antifungal resistance, and the mycotoxins and cytotoxicity profile. Total bacteria presented the highest prevalence in both matrixes, whereas Gram-bacteria presented the lowest. Swabs presented a higher prevalence (27.6%) for fungal burden. Chrysonilia sitophila presented the highest prevalence in swabs, whereas for EDCs, C. sitophila and Mucor sp. were the most prevalent. Concerning Aspergillus genera on swabs, section Flavi was the one with the highest prevalence (58.02%), whereas, for EDCs, section Versicolores was the only section observed (100%). Aspergillus section Fumigati was detected in 10 swabs and 7 EDC samples and Aspergillus section Versicolores was detected in one EDC sample. Fungal growth on azole-supplemented media was observed in eight EDC samples. No mycotoxins were detected in any of the samples. A low cytotoxic effect was observed in two sites upon incubation of collected samples with A549 and SK cells and in two other sites upon incubation of collected samples with SK cells only. A medium cytotoxic effect was observed with one EDC sample upon incubation with A549 cells. This study reinforces the need of determination of the azole resistance profile for fungal species and allowed a preliminary risk characterization regarding the cytotoxicity. An intervention including the use of a ultraviolet with wavelength between 200 nm and 280 nm (UVC)&mdash, emitting device and an increased maintenance and cleaning of the central heating, ventilation, and air conditioning (HVAC) systems should be ensured to promote the reduction of microbial contamination.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > C(T)) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 β-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 μg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log(10) bioburden reduction of 8% and 44% fT > 2 μg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.

Distal limb wounds are common injuries sustained by horses and their healing is fraught with complications due to equine anatomy, prevalence of infection, and challenges associated with wound management. Gallium is a semi-metallic element that has been shown to possess antimicrobial properties and aid in wound healing in various preclinical models. The effects of Gallium have not been studied in equine wound healing. Therefore, the objective of this study was to compare healing rates between gallium-treated and untreated wounds of equine distal limbs and to demonstrate the antimicrobial effects of gallium on wounds inoculated with S. aureus. Using an established model of equine wound healing we demonstrated beneficial effects of 0.5% topical gallium maltolate on equine wound healing. Specifically we documented reduced healing times, reduced bioburden, and reduced formation of exuberant granulation tissue in wounds treated with gallium maltolate as compared with untreated wounds. Gallium appeared to exert its beneficial effects via its well-described antimicrobial actions as well as by altering the expression of specific genes known to be involved in wound healing of horses and other animals. Specifically, gallium maltolate appeared to increase expression of transforming growth factor-β in both infected and un-infected wounds. Further work is needed to document the effects of gallium on naturally occurring equine wounds and to compare the effects of gallium with other wound treatment options. These data, however, suggest that gallium may be an attractive and novel means of improving equine distal limb wound healing.

The hygienic risk associated with evaporative cooling systems in Germany is currently only assessed by determining concentrations of Legionella spp. in the corresponding cooling waters. Relevant for the health risk is however the load of Legionella in emitted aerosols. In this work aerosol emissions from four industrial cooling systems (A - D) were analyzed. A microbiological air bioburden factor (MABF) is suggested to be useful to assess the overall microbiological load of emitted air and to judge the efficiency of droplet separation and overall microbiological retention. Whereas the MABF by itself only serves as a technical quality assurance (QA) parameter, the hygienic relevance has to be seen in combination with the assessment of Legionella either contained in the aerosol or in the cooling water. Plate counting of colonies was an appropriate method to quantify Legionella spp. in aerosols given the short time of flight at the chosen sampling locations and resulting low risk of desiccation. qPCR data on the other hand proved more reproducible than the culture approach to quantify Legionella spp. concentrations in cooling water-. The application of qPCR also allowed to assess the relative proportion of Legionella pneumophila within the total pool of Legionella which adds epidemiological relevance to risk assessment. A traffic light system was proposed to guide interpretation of qPCR data. The four industrial systems greatly differed in all measured parameters leading to different associated risks.

Pharmaceutical microbiology is the study of microorganisms such as bacteria and fungi present in or on the drug and pharmaceutical product, during manufacturing of injectable or non- injectable items these microorganisms enter in the preparation hence decrease the self-life of products or contaminate the product which cerate the problems in human being. In pharmaceutical industry water, raw material, intermediate products and finished products are checked for enumeration and isolation of microorganism. Presence and absence of common human pathogenic organisms such as Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella, Clostridia, Candida albicans, Bile-Tolerant Gram-Negative Bacteria etc. are checked. Pharmaceutical preparation is also received for other parameter like total aerobic microbial count and total yeast and mould count. Sterility test of parenteral and non- parenteral products are also done by using membrane filtration and direct inoculation techniques. In this chapters various methods such as estimation of antibiotic potency, vitamin B12 assay, bacterial endotoxin, quantification of calcium pantothenate and biotin, antimicrobial effectiveness testing, bioburden, determination of the level of lactic acid bacillus, Ames test, antimicrobial properties of pharmaceutically important compounds, multiple Antibiotic resistance and susceptibility pattern of microorganisms are described. Overall, in Drug and Pharmaceutical industry the products are manufactured in a controlled way as to produce the quality drugs which intended to provide the relief to mankind and society.

In bakeries, a number of operations such as mixing are associated with exposure to air-suspended flour dust and related bioburden. The aim of this study was to find the best active sampling approach to the assessment of occupational exposure to bioburden in Portuguese bakeries based on the data obtained with the use of specific impaction and impinger devices. We used impaction to collect fungal particles from 100 L air samples onto malt extract agar (MEA) supplemented with chloramphenicol (0.05 %). For growing fungi we also used dichloran glycerol (DG18) agar-based media and for mesophilic bacteria we used tryptic soy agar (TSA) supplemented with nystatin (0.2 %). For Enterobacteriaceae we used violet red bile agar (VRBA). With impingers we also collected 300 L air samples at the 300 L/min airflow rate, inoculated onto the same culture media. The two methods, impaction and impinger, showed statistically significant differences in the following counts: fungal on MEA (z=-2.721, p=0.007), fungal on DG18 (z=-4.830, p=0.000), total bacteria (z=-5.435, p=0.000), and Gram-negative coliforms (z=-3.716, p=0.000). In all cases the impaction method detected significantly higher concentrations than the impinger method. Fungal and bacterial loads were higher in the production unit and lower in the shop. The fungal load obtained with impaction varied between 10 and 5140 CFU m-3, and total bacterial counts ranged between 10 and 4120 CFU m-3. This study has shown that the impaction method is the best active sampling approach to assessing viable bioburden in this specific occupational environment, but a multi-faceted approach to sampling and analyses combining methods and media enables a more refined risk characterisation and, consequently, better tailored risk control measures to reduce adverse health outcomes in workers., Mnoge su djelatnosti u pekarama povezane s izloženosti lebdećim česticama brašna i pripadajućim mikroorganizmima, poput miješanja tijesta. Cilj je ovog istraživanja bio pronaći najbolji način aktivnog uzorkovanja radi ocjene profesionalne izloženosti biološkom opterećenju u portugalskim pekarama na temelju podataka dobivenih primjenom specifičnih impaktora i impingera. Impaktorom smo prikupljali čestice mikroskopski sitnih gljiva iz 100-litarskog uzorka zraka na agar od sladnog ekstrakta (engl. malt extract agar, krat. MAE) kojem je dodan kloramfenikol (0,05 %). Za rast gljiva također smo rabili dikloran glicerol (DG18) medij, a za mezofilne bakterije triptični sojin agar (TSA) s nistatinom (0,2 %). Za enterobakterije smo rabili ljubičasto-crveni žučni agar (engl. violet red bile agar, krat. VRBA). Impingerima smo također prikupljali uzorke zraka (300 L) uz protok od 300 L po minuti te ih cijepili na iste medije. Dvije su se metode, impakcijska i impingerska, statistički značajno razlikovale u sljedećim parametrima: u opterećenju gljivama na mediju MEA (z=-2,721, p=0,007), na mediju DG18 (z=-4,830, p=0,000), ukupnom bakterijskom opterećenju (z=-5,435, p=0,000) te u opterećenju gram-negativnim koliformima (z=-3,716, p=0,000). U svim je tim slučajevima impakcijska metoda dala značajno više koncentracije od impingerske. Opterećenje gljivama i bakterijama bilo je, sasvim očekivano, više u prostoriji za pripremu tijesta, a niže u prodavaonici. Opterećenje gljivama dobiveno impakcijom kretalo se u rasponu od 10 do 5140 CFU m-3, a bakterijama u rasponu od 10 do 4120 CFU m-3. Ovo je istraživanje izdvojilo impakcijsku metodu kao najbolji pristup aktivnom prikupljanju uzoraka za ocjenu opterećenja živim mikroorganizmima za ovaj specifični radni okoliš, ali bi višestrani pristup uzorkovanju i analizi, koji bi objedinjavao više metoda i medija, omogućio finiju karakterizaciju rizika, a time i mjere kontrole rizika koje bi bile bolje prilagođene specifičnim potrebama radi smanjenja štetnih zdravstvenih ishoda u radnika.

PURPOSE To elucidate the bioburden of a lens care system in patients with contact lens (CL)-related keratitis. METHODS In this prospective case-control study, the CL storage case was used as the target of bioburden assessment. Participants were CL wearers with (n = 26) or without (n = 24) keratitis in southern Taiwan. The case fluid sample was consecutively collected and assessed using a dot hybridization assay (DHA) for bioburden assessment. The DHA was composed of 3 universal bacterial probes for detecting all bacteria, genus-specific probes for Pseudomonas and Acinetobacter, and 1 probe for Acanthamoeba. The receiver operating characteristic curve was used to analyze the differentiating performance of a probe. RESULTS The storage case bioburden was significantly higher in those with CL-related keratitis than in control subjects; patients with CL-related keratitis had stronger standardized signals in all universal bacterial probes. Moreover, in the cases of these patients, the bioburden was significantly heavier in confirmed infectious keratitis than in presumed noninfectious keratitis. Receiver operating characteristic analysis showed that stronger signals in all 3 universal bacterial probes and the Pseudomonas probe might suggest that the wearers have infectious keratitis. Only the storage case of the Acanthamoeba keratitis case showed positive detection by the Acanthamoeba probe. CONCLUSIONS A heavier bioburden in the lens storage case was associated with a higher risk of CL-related keratitis and infectious keratitis. Inappropriate maintenance of the CL will lead to microbial contamination and transfer the pathogen onto the ocular surface causing keratitis accordingly. The DHA assessment for the lens storage case might provide an alternative way to differentiate infectious from noninfectious CL-related keratitis.

Endoscopy-associated infection transmission is frequently linked to inadequate reprocessing. Residual organic material and moisture may foster biofilm development inside endoscopes. This study evaluated the effectiveness of endoscope drying and storage methods and assessed associations between retained moisture and contamination.Endoscope reprocessing, drying, and storage practices were assessed at 3 hospitals. Researchers performed visual examinations and tests to detect fluid and contamination on patient-ready endoscopes.Fluid was detected in 22 of 45 (49%) endoscopes. Prevalence of moisture varied significantly by site (5%; 83%; 85%; P .001). High adenosine triphosphate levels were found in 22% of endoscopes, and microbial growth was detected in 71% of endoscopes. Stenotrophomonas maltophilia, Citrobacter freundii, and Lecanicillium lecanii/Verticillium dahliae were found. Retained fluid was associated with significantly higher adenosine triphosphate levels (P .01). Reprocessing and drying practices conformed with guidelines at 1 site and were substandard at 2 sites. Damaged endoscopes were in use at all sites.Inadequate reprocessing and insufficient drying contributed to retained fluid and contamination found during this multisite study. More effective methods of endoscope reprocessing, drying, and maintenance are needed to prevent the retention of fluid, organic material, and bioburden that could cause patient illness or injury.

Early awareness and management of bacterial burden and biofilm is essential to wound healing. Semi-quantitative analysis of swab or biopsy samples is a relatively simple method for measuring wound microbial load. The accuracy of semi-quantitative culture analysis was compared to ‘gold standard’ quantitative culture analysis using 428 tissue biopsies from 350 chronic wounds. Semi-quantitative results, obtained by serial dilution of biopsy homogenates streaked onto culture plates divided into 4 quadrants representing occasional, light, moderate, and heavy growth, were compared to total bacterial load quantified as colony-forming units per gram (CFU/g). Light growth, typically considered an insignificant finding, averaged a clinically significant 2.5 × 105 CFU/g (SE = 6.3 × 104 CFU/g). Occasional growth (range: 102–106 CFU/g) and light growth (103–107 CFU/g) corresponded to quantitative values that spanned a 5-log range, moderate and heavy growth corresponded to a range of 4-log and 6-log, respectively, with a high degree of overlap in range of CFU/g per category. Since tissue biopsy and quantitative culture cannot be widely practiced and semi-quantitative analysis is unreliable, other clinically relevant approaches are required to determine wound bioburden and guide best management practices. Fluorescence imaging is a point-of-care technology that offers great potential in this field.

Background The ExoMars 2016 mission, consisting of the Trace Gas Orbiter and the Schiaparelli lander, was launched on March 14 2016 from Baikonur, Kazakhstan and reached its destination in October 2016. The Schiaparelli lander was subject to strict requirements for microbial cleanliness according to the obligatory planetary protection policy. To reach the required cleanliness, the ExoMars 2016 flight hardware was assembled in a newly built, biocontrolled cleanroom complex at Thales Alenia Space in Turin, Italy. In this study, we performed microbiological surveys of the cleanroom facilities and the spacecraft hardware before and during the assembly, integration and testing (AIT) activities. Methods Besides the European Space Agency (ESA) standard bioburden assay, that served as a proxy for the microbiological contamination in general, we performed various alternative cultivation assays and utilised molecular techniques, including quantitative PCR and next generation sequencing, to assess the absolute and relative abundance and broadest diversity of microorganisms and their signatures in the cleanroom and on the spacecraft hardware. Results Our results show that the bioburden, detected microbial contamination and microbial diversity decreased continuously after the cleanroom was decontaminated with more effective cleaning agents and during the ongoing AIT. The studied cleanrooms and change room were occupied by very distinct microbial communities: Overall, the change room harboured a higher number and diversity of microorganisms, including Propionibacterium, which was found to be significantly increased in the change room. In particular, the so called alternative cultivation assays proved important in detecting a broader cultivable diversity than covered by the standard bioburden assay and thus completed the picture on the cleanroom microbiota. Conclusion During the whole project, the bioburden stayed at acceptable level and did not raise any concern for the ExoMars 2016 mission. The cleanroom complex at Thales Alenia Space in Turin is an excellent example of how efficient microbiological control is performed. Electronic supplementary material The online version of this article (10.1186/s40168-017-0358-3) contains supplementary material, which is available to authorized users.

Combat injuries are associated with a high incidence of infection, and there is a continuing need for improved approaches to control infection and promote wound healing. Due to the possible local and systemic adverse effects of standard 1% cream formulation (Silvadene), we had previously developed a polyethylene glycol (PEGylated) fibrin hydrogel (FPEG)-based wound dressing for the controlled delivery of silver sulfadiazine (SSD) entrapped in chitosan microspheres (CSM). In this study, we have evaluated the antimicrobial and wound healing efficacy of SSD-CSM-FPEG using a full-thickness porcine wound infected with Pseudomonas aeruginosa. Infected wounds treated with a one-time application of the SSD-CSM-FPEG wound dressing demonstrated significantly reduced bacterial bioburden over time (99·99% of reduction by day 11; P < 0·05) compared with all the other treatment groups. The epithelial thickness and granulation of the wound bed was significantly better on day 7 (150·9 ± 13·12 µm), when compared with other treatment groups. Overall, our findings demonstrate that the SSD-CSM-FPEG wound dressing effectively controls P. aeruginosa infection and promotes wound healing by providing a favourable environment that induces neovascularisation. Collectively, sustained release of SSD using fibrin hydrogel exhibited enhanced benefits when compared with the currently available SSD treatment, and this may have significant implications in the bacterial reduction of infected wounds in military and civilian populations.

This study investigated whether there are differences in the ability of wound dressings to modulate certain factors known to affect wound healing. A selection of antimicrobial dressings (AQUACEL® Ag Extra™ , AQUACEL® Ag+ Extra™ , IODOFLEX™ , ACTICOAT™ 7 and PROMOGRAN PRISMA™ matrix) were tested for their effect on both bacterial bioburden and human dermal fibroblasts. Some dressings underwent further evaluation for activity against Pseudomonas aeruginosa biofilms using a colony-drip flow reactor model. The ability of in vitro biofilms to produce proteases, and the effect of PROMOGRAN PRISMA matrix on such proteases, was also investigated. All antimicrobial dressings tested reduced vegetative bacterial load; however, only PROMOGRAN PRISMA matrix was able to significantly reduce biofilm populations (P = 0·01). Additionally, PROMOGRAN PRISMA matrix was the only dressing that did not inhibit dermal fibroblast growth. All other dressings were detrimental to cell viability. In vitro biofilms of Pseudomonas aeruginosa were demonstrated as being capable of releasing bacterial proteases into their surroundings, and incubation with PROMOGRAN PRISMA matrix led to a 77% reduction in activity of such proteases (P = 0·002). The unique ability of PROMOGRAN PRISMA matrix to reduce in vitro vegetative bacteria, biofilm bacteria and bacterial proteases while still allowing dermal fibroblast proliferation may help rebalance the wound environment and reduce the occurrence of infection.

OBJECTIVETo evaluate the efficacy of copper-coating in reducing environmental colonization in an intensive-care unit (ICU) with multidrug-resistant-organism (MDRO) endemicityDESIGNInterventional, comparative crossover trialSETTINGThe general ICU of Attikon University hospital in Athens, GreecePATIENTSThose admitted to ICU compartments A and B during the study periodMETHODSBefore any intervention (phase 1), the optimum sampling method using 2 nylon swabs was validated. In phase 2, 6 copper-coated beds (ie, with coated upper, lower, and side rails) and accessories (ie, coated side table, intravenous [i.v.] pole stands, side-cart handles, and manual antiseptic dispenser cover) were introduced as follows: During phase 2a (September 2011 to February 2012), coated items were placed next to noncoated ones (controls) in both compartments A and B; during phase 2b (May 2012 to January 2013), all copper-coated items were placed in compartment A, and all noncoated ones (controls) in compartment B. Patients were randomly assigned to available beds. Environmental samples were cultured quantitatively for clinically important bacteria. Clinical and demographic data were collected from medical records.RESULTSCopper coating significantly reduced the percentage of colonized surfaces (55.6% vs 72.5%; PP=.003) or by enterococci (4% vs 17%; P=.014), the total bioburden (2,858 vs 7,631 cfu/100 cm2; P=.008), and the bioburden of gram-negative isolates, specifically (261 vs 1,266 cfu/100 cm2; P=.049). This effect was more pronounced when the ratio of coated surfaces around the patient was increased (phase 2b).CONCLUSIONSCopper-coated items in an ICU setting with endemic high antimicrobial resistance reduced environmental colonization by MDROs.Infect Control Hosp Epidemiol 2017;38:765–771

Background: The aim of this study was to measure the changes in the bacterial bioburden in orthokeratology (OK) lens storage cases using the DNA dot hybridization assay (DHA) after forewarning patients about their bacterial contamination severity. Methods: Thirty-one OK lens wearers were prospectively enrolled in this study. Dot hybridization assay was used for serial measurements of bacterial bioburden in OK storage cases after lenses had been soaked for approximately 6 hr. After the first assessment, the lens wearers were informed of the extent of case contamination and the possible risk of microbial keratitis (MK), and best practices for lens care and lens case hygiene were reviewed and reinforced. A second assessment by the same DHA method was performed after approximately 6 months. Results: Two universal bacterial probes confirmed a significant decrease in bacterial bioburden at the second assessment (P

Background and objectives Bacterial biofilm formation within chronic wound beds, which provides an effective barrier against antibiotics, is a known cause of recalcitrant infections and a significant healthcare burden, often requiring repeated surgical debridements. Laser-generated shockwaves (LGS) is a novel, minimally invasive, and nonthermal modality for biofilm mechanical debridement which utilizes compressive stress waves, generated by photonic absorption in thin titanium films to mechanically disrupt the biofilm. Prior studies have demonstrated LGS monotherapy to be selectively efficacious for biofilm disruption and safe for host tissues. In this study, we sought to determine if LGS can enhance the antimicrobial activity and biofilm disruption capability of topical antibiotic therapy. Study design/materials and methods Staphylococcus epidermidis biofilms grown in vitro on glass were treated with topical gentamicin (31, 62, and 124 μg/ml) with and without LGS (n = 3-11/treatment group). Mechanical shockwaves were generated with a 1,064 nm Nd:YAG laser (laser fluence 110.14 mJ/mm2 , pulse duration 5 ns, spot size 3 mm). Following a 24-hour incubation period, bacterial viability was assessed by determining the number of colony-forming units (CFU) via the Miles and Misra method. Residual biofilm bioburden was analyzed using the crystal violet biofilm assay. Results With gentamicin monotherapy, CFU density (CFU/mm2 ) at 31, 62, and 124 μg/ml were (282 ± 84) × 104 , (185 ± 34) × 104 , and (113 ± 9) × 104 , respectively. With LGS and gentamicin therapy, CFU density decreased to (170 ± 44) × 104 , (89 ± 24) × 104 , and (43 ± 3) × 104 , respectively (P = 0.1704, 0.0302, and 0.0004 when compared with gentamicin alone). Biofilm burden as measured by the assay in the gentamicin 31, 62, and 124 μg/ml groups was reduced by 80%, 95%, and 98% when LGS was added (P = 0.0102, >0.0001, and 0.0001 for all groups when compared with gentamicin alone). Furthermore, samples treated with LGS saw an increase in susceptibility to gentamicin, in terms of reduced biofilm bioburden and CFU densities. Conclusion LGS enhances the efficacy of topical antibiotics in an in vitro model. This has significant implications for clinical applications in the management of chronic soft tissue infections and recalcitrant chronic rhinosinusitis. Lasers Surg. Med. 49:539-547, 2017. © 2017 Wiley Periodicals, Inc.

Prevention measures are crucial in actual production, whereas the outbreak of a disease requires immediate detection and elimination of the source of infection as well as complex veterinary and sanitary measures. Here the critical role is attributed to disinfection, which breaks the epizootic chain due to the elimination of pathogenic microorganisms on the objects under veterinary surveillance and involves the application of various disinfectants. Aim. The study of virucidal properties of the novel disinfectant DZPT-2. Methods. The culture technique was used at the fi rst stage of studies to determine the embryotoxic effect of the preparation on chicken embryos and the cytotoxic effect of the disinfectant on BHK-21 cell culture. The second stage of experiments envisaged the study of virucidal effect of the disinfectant using both the suspension method, test- objects (batiste, wood, glazed tile, metal, glass) and the bioburden. Results. The experiments using the agent of bovine viral diarrhea demonstrated that the disinfectant DZPT-2 in the concentration of 1.0 % of the active ingredient (AI) when exposed for at least 30 min and in the concentration of 1.5 % of AI when exposed for 15– 60 min disinfects all the test-objects, contaminated by the virus, completely. When exposed for up to 15 min, the disinfectant DZPT-2 does not demonstrate its virucidal effect on Newcastle disease virus, but it disinfects all the test-objects, contaminated by the mentioned virus, when used in the concentration of 0.5–1.0 % of AI and exposed for at least 30 min. Conclusions. It was determined that the preparation DZPT-2 demonstrates its virucidal properties in the concentration of 0.5 % of AI (Newcastle disease virus) and 1.0 % of AI (agent of bovine viral diarrhea) when exposed for 30 min. The new disinfectant DZPT-2 is a promising preparation to be used in practical veterinary medicine to prevent and fi ght viral diseases of farm livestock and poultry.

Numerous cases have been reported globally showing outbreaks, dissemination of infections and/or spoilage of medicinal products, food and other consumables that affects human life and may lead to death in some cases. One of the most critical measures of microbial contamination that should be taken into consideration is the use of proper disinfectant depending on the type of activities and the work load of the healthcare institution, subjects or media for microbial transfer and the affected population. The current study shows significant difference in sporicidal activity between two types of commercial peroxygen/Silver based disinfectants obtained from same manufacturer and delivered through same distributor at the same time. The peroxygen component in one of the disinfectant products is Hydrogen peroxide while in the other product is Peroxyacetic acid/Hydrogen Peroxide mixture. Preliminary rapid assessment of the disinfectants activity was required using the most resistance microbial form (bacterial spore) as a reference microorganism to challenge the biocidal products. Bacillus pumilus and B. subtilis spores were exposed to both disinfectants at three different concentrations levels covering the range recommended by the manufacturer: 1, 3 and 5% (v/v). The first formula did not exceed 0.5 logarithmic reduction(LR) even after 30 minutes. While the other product achieved more than two folds LR (more than 100 times reduction in microbial population) after ten minutes contact time. Appropriate initial screening of biocidal activity in commercial disinfectants market is critical step that should be performed by the healthcare facilities before practical application. Otherwise, inefficient control on bioburden may lead to devastating consequences on human health. An initial, non-laborious, time-saving and non-expensive screening test using the most resistance microorganisms is encouraged to be performed by healthcare facilities prior to practical application of disinfectant rather than reliance solely on random selection of biocidal agents using informational data without confirmatory experiments.

The bioburden in a Hospital building originates not only from patients, visitors and staff, but is also disseminated by several indoor hospital characteristics and outdoor environmental sources. This study intends to assess the exposure to bioburden in one central Hospital with a multi-approach protocol using active and passive sampling methods. The microbial contamination was also characterized through molecular tools for toxigenic species, antifungal resistance and mycotoxins and endotoxins profile. Two cytotoxicity assays (MTT and resazurin) were conducted with two cell lines (Calu-3 and THP-1), and in vitro pro-inflammatory potential was assessed in THP-1 cell line. Out of the 15 sampling locations 33.3% did not comply with Portuguese legislation regarding bacterial contamination, whereas concerning fungal contamination 60% presented I/O > 1. Toxigenic fungal species were observed in 27% of the sampled rooms (4 out of 15) and qPCR analysis successfully amplified DNA from the Aspergillus sections Flavi and Fumigati, although mycotoxins were not detected. Growth of distinct fungal species was observed on Sabouraud dextrose agar with triazole drugs, such as Aspergillus section Versicolores on 1 mg/L VORI. The highest concentrations of endotoxins were found in settled dust samples and ranged from 5.72 to 23.0 EU.mg-1. While a considerable cytotoxic effect (cell viability

We studied the association between chlorhexidine gluconate (CHG) concentration on skin and resistant bacterial bioburden. CHG was almost always detected on the skin, and detection of methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant Enterococcus on skin sites was infrequent. However, we found no correlation between CHG concentration and bacterial bioburden.

Antimicrobial resistance has been recognized as a threat to human health. The role of hospital sinks acting as a reservoir for some of the most concerning antibiotic resistant organisms, carbapenemase producing Enterobacterales (CPE) is evident but not well understood. Strategies to prevent establishment, interventions to eliminate these reservoirs and factors which drive persistence of CPE are not well established. We use a uniquely designed sink lab to transplant CPE colonized hospital sink plumbing with an aim to understand CPE dynamics in a controlled setting, notably exploiting both molecular and culture techniques. After ex situ installation the CPE population in the sink plumbing drop from previously detectable to undetectable levels. The addition of nutrients is followed by a quick rebound in CPE detection in the sinks after as many as 37 days. We did not however detect a significant shift in microbial community structure or the overall resistance gene carriage in longitudinal samples from a subset of these transplanted sinks using whole shotgun metagenomic sequencing. Comparing nutrient types in a benchtop culture study model, protein rich nutrients appear to be the most supportive for CPE growth and biofilm formation ability. The role of nutrients exposure is determining factor for maintaining a high bioburden of CPE in the sink drains and P-traps. Therefore, limiting nutrient disposal into sinks has reasonable potential with regard to decreasing the CPE wastewater burden, especially in hospitals seeking to control an environmental reservoir.

Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and –negative bacteria.

One of the solutions for decreasing the workers' exposure to bioburden is the use of Filtering Respiratory Protective Devices (FRPD). As such it is important to determine whether these devices are fulfilling their protective role. This is the basis of the current study, aimed at characterizing bioburden retained by 120 FRPD (both in interior layers and in exhalation valves) through culture based-methods and molecular tools and also via analysis of antifungal resistance and mycotoxins profile. Our results show that Gram – Bacteria are present at a higher prevalence than total bacteria in both matrixes. Regarding fungal identification, Chrysonilia sitophila presented the highest prevalence on interior layers (55.1% on malt extract agar (MEA) supplemented with chloramphenicol (0.05%); 59.6% on dichloran-glycerol agar (DG18)), whereas on exhalation valves Aspergillus sp. presented the highest prevalence on MEA (6.8%) and C. sitophila on DG18 (36.3%). Among Aspergillus genera, section Fumigati was the one with the highest prevalence in both matrices. Aspergillus sp. was the most prevalent on exhalation valves (75.0% ITRA) in the screening of azole resistance. Fumigati section was the most abundant Aspergillus sp. detected on the interior layers (33.33%, 40 samples out of 120) and on the exhalation valves (1.66%, 2 samples out of 120). The interior layers and exhalation valves from workers with more waste contact showed an increased exposure to bioburden. This study showed that FRPD can have high levels of bioburden, toxigenic fungal strains and Aspergillus sections with reduced susceptibility to the tested azoles and can be used as a passive sampling method since it mimics the results obtained by active methods in previous studies. The gathered information will be useful to prioritize multiple interventions on workers’ education or even on FRPD replacement frequency.

The efficacy of domestic laundering of healthcare staff clothing is still debated. This study aimed to compare the performance of decontamination of different domestic laundering with that of industrial laundering. Fourteen naturally contaminated white coats of healthcare workers (5 fabric squares from each coat) and fabric squares of artificially contaminated cotton cloth (30 fabric squares per each bacterial strain used) were included. Four domestic laundering procedures were performed; two different washing temperatures (40°C and 90°C) and drying (tumble dry and air dry) were used. All fabric squares were ironed. Presence of bacterial bioburden on the fabric squares after domestic and industrial laundering was investigated. None of the naturally contaminated fabric squares resulted completely decontaminated after any of the domestic washes. At 24, 48, and 72h of incubation, bacterial growth was observed in all the laundered fabric squares. Besides environmental microorganisms, potentially pathogenic bacteria (i.e., Acinetobacter lwoffii, Micrococcus luteus, coagulase-negative staphylococci) were isolated. On the artificially contaminated fabric squares, the bioburden was reduced after the domestic laundries; nevertheless, both Gram-negative and -positive pathogenic bacteria were not completely removed. In addition, a contamination of the fabric squares by environmental Gram-negative bacteria was observed. In both the naturally and artificially contaminated fabric squares, no bacterial growth at all the time-points analyzed was observed after industrial laundering, which provided to be more effective in bacterial decontamination than domestic washes. For those areas requiring the highest level of decontamination, the use of specialized industrial laundry services should be preferred.

BackgroundEvidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices.Materials and methodsIn vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition.ResultsIt was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium).ConclusionsOverall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

This study aimed to evaluate the sanitary status of fresh bovine meat sold at the open market in Jiquiriça municipality, Brazil and validate the use of chitosan as an antimicrobial coating to control the bioburden. In total, we evaluated 30 samples of fresh beef for the presence of coagulase-positive staphylococci and Salmonella spp. Furthermore, stores selling fresh meat products were evaluated for compliance with sanitary regulations set by the Ministry of Health in Brazil. To evaluate the antimicrobial activity of chitosan two sets of meat samples were tested, the test group consisted of contaminated meat coated with 2% chitosan solution whereas the control group consisted of untreated contaminated meat. It was found that fresh bovine meat at market stores is exposed to a number of contaminants, including several pathogens. All of the tested samples contained coagulase-positive staphylococci (3.8-6.1 log CFU g-1) and Salmonella spp. were detected in 30% of all samples. When assessing the sanitary conditions of the meat stores, non-compliance was highest in the marketing and display category (79%), followed by the hygiene of manipulators (73%) and the facilities, equipment and utensils categories (54%). Meat treated treated with 2% chitosan solution showed significant lower (p

The primary adventitious agents of concern for biologics are examined in detail—prions, viruses, mycoplasmas, and bacteria/fungi microbes. The three major risk control procedures for these contaminating agents—barriers to entry, testing to confirm absence, and inactivation/removal—are discussed. Risk control examples are provided for animal-derived materials used in biologic manufacturing, cell culture manufacturing processes, biopharmaceutical production in transgenic animals and plants, natural sourced human plasma-derived proteins, virus-based biologics, and cell-based biologic medicines. In addition, lessons learned from previously reported infectious agent contaminations of biologics are presented. Finally, some CMC strategic tips are provided for minimizing the overall risk of adventitious agent contamination of biologics.

ObjectiveTo determine the efficacy of 2 types of antimicrobial privacy curtains in clinical settings and the costs involved in replacing standard curtains with antimicrobial curtains.DesignA prospective, open-labeled, multicenter study with a follow-up duration of 6 months.SettingThis study included 12 rooms of patients with multidrug-resistant organisms (MDROs) (668 patient bed days) and 10 cubicles (8,839 patient bed days) in the medical, surgical, neurosurgical, orthopedics, and rehabilitation units of 10 hospitals.MethodCulture samples were collected from curtain surfaces twice a week for 2 weeks, followed by weekly intervals.ResultsWith a median hanging time of 173 days, antimicrobial curtain B (quaternary ammonium chlorides [QAC] plus polyorganosiloxane) was highly effective in reducing the bioburden (colony-forming units/100 cm2, 1 vs 57; P < .001) compared with the standard curtain. The percentages of MDRO contamination were also significantly lower on antimicrobial curtain B than the standard curtain: methicillin-resistant Staphylococcus aureus, 0.5% vs 24% (P < .001); carbapenem-resistant Acinetobacter spp, 0.2% vs 22.1% (P < .001); multidrug-resistant Acinetobacter spp, 0% vs 13.2% (P < .001). Notably, the median time to first contamination by MDROs was 27.6 times longer for antimicrobial curtain B than for the standard curtain (138 days vs 5 days; P = .001).ConclusionsAntimicrobial curtain B (QAC plus polyorganosiloxane) but not antimicrobial curtain A (built-in silver) effectively reduced the microbial burden and MDRO contamination compared with the standard curtain, even after extended use in an active clinical setting. The antimicrobial curtain provided an opportunity to avert indirect costs related to curtain changing and laundering in addition to improving patient safety.

Background Ultraviolet (UV) spectrum light for decontamination of patient care areas is an effective way to reduce transmission of infectious pathogens. Our purpose was to investigate the efficacy of an automated UV-C device to eliminate bioburden on hospital computer keyboards. Methods The study took place at an academic hospital in Chicago, Illinois. Baseline cultures were obtained from keyboards in intensive care units. Automated UV-C lamps were installed over keyboards and mice of those computers. The lamps were tested at varying cycle lengths to determine shortest effective cycles. Delay after use and prior to cycle initiation was varied to minimize cycle interruptions. Finally, 218 postinstallation samples were analyzed. Results Of 203 baseline samples, 193 (95.1%) were positive for bacteria, with a median of 120 colony forming units (CFU) per keyboard. There were numerous bacteria linked to health care–associated infections (HAIs), including Staphylococcus , Streptococcus , Enterococcus , Pseudomonas , Pasteurella , Klebsiella , Acinetobacter , and Enterobacter . Of the 193 keyboards, 25 (12.3%) had gram-negative species. Of 218 postinstallation samples, 205 (94%) were sterile. Of the 13 that showed bacterial growth, 6 produced a single CFU. Comparison of pre- and post-UV decontamination median CFU values (120 and 0, respectively) revealed a >99% reduction in bacteria. Conclusions The UV lamp effectively decontaminates keyboards with minimal interruption and low UV exposure. Further studies are required to determine reduction of HAI transmission with use of these devices.

Bacterial contamination control in pharmaceutical products is a critical aspect in the field of drug manufacturing industry due to the encountered risk to the patients' health and possibly their life. The application of commercial bacterial identification system is crucial to identify the type of contamination and its source to anticipate the impact of bioburden on the products and setting corrective and preventive actions. During the period of one year, random samples from raw materials and final products were tested according to United States Pharmacopeia, and those that showed suspect results for specified microorganisms and/or out-of-specification limits or showed out-of-trend results were subjected to further identification by using miniaturized biochemical identification system after performing Gram stain. From the total bacterial isolates of the investigated products, more than 60% were primarily belonging to Micrococcaceae 16.98% (empty hard gelatin capsules), Enterobacteriaceae 18.86% (vaginal cream applicator, plastic caps for bottles, Sorbitol solution, finished hard gelatin capsule product, topical cream and oral suspension) and Bacillaceae 24.53% (Talc powder, liquid oral preparation and finished hard gelatin capsule product). Gram Positive and Negative samples were 56.60% and 41.51% respectively from the total investigated sample products and materials. Finished pharmaceutical products constituted 53.33% and 68.18% from Gram-positive and Gram-negative microorganisms respectively. An approach to quantitative risk assessment for pharmaceutical products was conducted on selected medicinal items and showed that Enterobacteriaceae followed by Burkholderiaceae contributed by more than 80% to the major hazard that could be delivered to patients through drugs. The applied risk can be used as a milestone for setting goals by pharmaceutical companies to improve the safety of medicinal products microbiologically and to identify the major sources of the risk to work on it in order to deliver safe drugs to the customers.

Cardiovascular allografts are usually disinfected using antibiotics, but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of efficacy; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. We conducted a systematic review of methods applied to disinfect cardiovascular tissues. The use of multiple broad spectrum antibiotics in conjunction with an antifungal agent resulted in the greatest reduction in bioburden. Antibiotic incubation periods were limited to less than 24 h, and most protocols incubated tissues at 4 °C, however one study demonstrated a greater reduction of microbial load at 37 °C. None of the reviewed studies looked at the impact of these disinfection protocols on the risk of infection or any other clinical outcome in recipients. Electronic supplementary material The online version of this article (doi:10.1007/s10561-016-9570-9) contains supplementary material, which is available to authorized users.