Environmental Microbiology

Environmental Microbiology

Tuesday, June 2, 2015, 10:30 AM - 12:30 PM

SR-112-01 Characterization of a Piezoelectric Inkjet Aerosol Generator f​​​or Bioaerosol Survivability Study

Y. Kuo, Chung Hwa University of Medical Technology, Tainan, Taiwan; S. Huang, C. Lin, W. Wu, W. Ke, C. Chen, National Taiwan University, Taipei, Taiwan

Objective: For a laboratory-based bioaerosol research, stable and reliable bioaerosol generation with minimal damage to microorganisms is of great importance. The main objective of this study is to characterize a piezoelectric inkjet aerosol generator for bioaerosol survivability study.

Methods: The aerosol generation system consists of a 50-μm piezoelectric print head, a function generator, a power amplifier, and a pressure control device. The performance of this system is characterized using the potassium sodium tartrate solution and the 3-µm polystyrene latex suspension. The main operating parameters of the inkjet print head for viable aerosol generation include driving voltage, pulse duration, dispersion air flow, pulse frequency and volume fraction of solids in the nebulizing solution. For bioaerosol survival experiments, yeast is used as the test microorganism. Three bioaerosol samplers including a BioSampler, a cassette with gelatine filter and a Marple cascade impactor with Mylar film are used to collect airborne yeast samples. The yeast survival is then determined by methylene blue viable staining with microscopy.

Results: This study successfully demonstrates the feasibility and the merit of using piezoelectric inkjet print head for generating viable aerosols. A square wave with appropriate combinations of voltage and pulse duration can drive the inkjet system to squeeze liquid with rheology characteristic similar to water. The particle generation rate can be manipulated by adjusting the pulse frequency. Moreover, dispersion air is required to reduce coagulation. The size distribution of particle dried from 3 mm PSL suspension shows two distribution peaks. The smaller one is apparently from the soluble residue of suspension. The larger one represents the size distribution of particles dried from main droplets. The appropriate concentration of solid suspension is in the magnitude of 106 #/ml. The fraction of droplets containing at least one particle is 0.26. The fraction of droplets containing only one particle among droplets containing at least one particle is 0.86.

Conclusions: The inkjet-based aerosol generation has been demonstrated to be more controllable, dynamic, and reproducible than other generators. Moreover, piezoelectric inkjet printing is a thermally consistent process and therefore the microbial survival could be expected to be well preserved.

SR-112-02 Transport of Microbial Particles on Fibers in Occupational Environments

R. Gorny, A. Lawniczek-Walczyk, A. Stobnicka, Central Institute for Labour Protection – National Research Institute, Warsaw, Poland

Objective: To check an ability of different natural and man-made fibers to transport microbial particles at different workplaces.

Methods: The sampling was carried out at: three industrial facilities processing natural (cotton, silk, flax, hemp, wool) and synthetic (polyamide, polyester, polyacrylonitrile, polypropylene, viscose) fibrous materials, one stable where horses were bred and one zoo pavilion where lions were kept. At each of these facilities, fibrous aerosol was sampled using 37-mm open-faced cassette on sterile teflon filter during “routine” activities, i.e. during final stages of fibrous material manufacturing cycle, horse currying and lion grooming. Simultaneously with aerosol sampling, settled fibrous dust was collected using sterile spatula. All man-made and natural fiber samples were weighed and laboratory analyzed for their microbiological purity. All microbial strains were taxonomically identified based on their morphology, biochemical features and identification keys. To check the structure and arrangement of fibers that may support transport of microbial particles, a scanning electron microscopy analysis was performed.

Results: Both airborne and settled fibers transported analogous microbial strains. All synthetic fibers, present as separated threads with smooth surface, were free from microbial contamination. Natural fibers with loose packing and rough surface (i.e. wool, horse hair, lion fur), sheaf packing and septated surface (e.g. flax, hemp) or present as twisted ribbons with corrugated surface (cotton) were able to carry up to 9×105/3.4×104 and 6.3×104 cfu/g of aerobic/anaerobic bacteria and fungi, respectively. The microbiota present on natural fibers comprised wide spectrum of species (from endospore-forming and non-sporing Gram-positive rods, through Gram-positive cocci, Gram-negative rods, to molds and yeasts), including pathogenic strains classified by Directive 2000/54/EC to hazard group 2.

Conclusions: As both plant and animal fibers transport a significant number of microbial particles, including pathogens, all of them should be thoroughly eliminated from workplaces and, if production cycle allows such a change, replaced by synthetic ones. To restrain unwanted presence, dissemination and subsequent exposure to harmful microbial agents, high-performance containment measures should be introduced during fiber material manufacturing.

SR-112-03 Seven Novel Penicillium Species and Two Novel Citreoviridin Producing Species of Section Eupenicillium

Z. Jurjevic, EMSL Analytical, Inc., Cinnaminson, NJ; S. Peterson, Microbial Genomics and Bioprocessing Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, Peoria, IL; J. Frisvad, Technical University of Denmark, Lyngby, Denmark

Objective: A set of isolates were obtained that did not fit any described species. Phenotypic examination of these isolates showed that they were very similar but differed in some growth characteristics. Multilocus DNA sequence data was obtained for the new isolates and some related species in the broader, more inclusive clade.

Methods: Fungal isolates were collected from air samples (home/factory) in several widely separated states of the USA and Denmark, from a sample of corn meal (South Africa), from pecan shell (USA, Georgia) and unknown origin. To describe the species we used phenotypes from 7 and 14 day Czapek’s yeast extract agar (CYA) and malt extract agar (MEA). Additional media were CYA with 20% sucrose (CY20S), and CYA supplemented with 5% NaCl, oatmeal agar (OA), and potato dextrose agar (PDA).This was followed by multilocus DNA sequence beta tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID), minichromosome maintenance complex component 7 (Mcm7), second largest subunit RNA polymerase (RPB2), and pre-rRNA processing protein (Tsr1) loci.

Results: Based on molecular, morphological and chemical analysis the new isolates are described as Penicillium colei, P. cvjetkovicii, P. fluviserpens, P. lehmiflumine, P. monsgalena, P. monsserratidens, and P. salmoniflumine. Extrolite analysis showed that two of the new species, P. colei and P. monsserratidens produce the mycotoxin citreoviridin.

Conclusions: The seven new species represent a monophyletic clade that also includes Penicillium idahoense. Citreoviridin or citreoviridin A is a neurotoxic mycotoxin that was first isolated from yellow rice and is believed to be the cause of acute cardiac beriberi disease in humans. Citreoviridin contaminated commodities are uncommon but acute cardiac beriberi was recently reported in Brazil and associated with moldy rice. Citreoviridin has been reported as a contaminant of maize and pecans in the USA. Some citreoviridin derivatives have shown pharmacological activity against lung tumors and these isolates could provide leads in pharmaceutical research. Occupational and environmental hygiene professionals will be made aware of these newly described fungi and their potential health hazards.

SR-112-04 Estimating the Accuracy of Single Outdoor Samples

H. Burge, Environmental Microbiology Laboratory, San Bruno, CA

Objective: Statistically, the indoor aerosol (by taxon) is well correlated to the outdoor aerosol with concentrations lower indoors. Thus, outdoor samples are essential to control for this outdoor/indoor penetration effect. Indoor/outdoor samples are usually collected within a short period of time while outdoor penetration has probably occurred before the samples are collected. Acute changes in weather could cause the outdoor sample data to misrepresent the actual prevailing aerosol that penetrated indoors. Our objective is to provide summarized baseline data specific to the date and location of the outdoor sample that can be used to evaluate the accuracy of single outdoor samples.

Methods: We have been mapping the outdoor fungal aerosol for many years, and have developed the MoldRange to assist investigators in interpreting outdoor sampling data. Beginning with monthly and by State averages separately then together, we went on to establish zip code groupings to more accurately represent fungal populations. At least for California, we are now able to offer minimum, median, and maximum data to our clients for comparison with their sample data for that date. This year we have expanded this effort and developed a preliminary excel spreadsheet that will allow a zip code and date to be entered and the computer will produce the range, minimum, median and maximum counts for that zip code. The statistic uses NOAA climate data on temperature and precipitation within the entered zip code and our massive database on outdoor fungal aerosols to generate these values.

Results: Preliminary results indicate that the statistic produces data within the range of the MOLDRANGE. It also provides average temperature and precipitation for the month of sampling by site, and the frequency of recovery of specific taxa at the site. It is adaptable for all locations for which NOAA provides weather data, and for which we have sufficient samples for the location in our database.

Conclusions: This research is providing a tool to evaluate the accuracy of single outdoor samples by comparing the sample data to our database by zip code, date, and weather parameters. In addition to its use for interpreting single samples, this statistic may also be used to track relationships between specific aerosols by temperature, precipitation and geographical area, and possibly even to help predict future aerosol patterns.

SR-112-05 Diseases Attributable to Common Indoor Molds

H. Burge, EMLab, San Bruno, CA

Objective: Molds are abundant in both outdoor and indoor air, with the vast majority of exposure occurring outdoors. However, indoor growth presents the possibility of exposure to higher concentrations of spores and other materials associated with fungal growth. We have reviewed the current peer-reviewed literature focusing on diseases caused by fungi that are commonly recovered from air and surfaces in the indoor environment.

Methods: The Web of Science was used for the literature search through a link with the University of Michigan. Only peer-reviewed literature was used, and conference proceedings were not included even if peer-reviewed. The focus was on the past 10 years (2004–2014), and approximately 150 papers were finally a part of this review.

Results: Penicillium, Aspergillus, Cladosporium and Alternaria are commonly isolated from indoor air, while Penicillium, Aspergillus, Stachybotrys, Acremonium and Chaetomium are common on surfaces. All may cause sensitization in susceptible people. Most sensitizing exposure occurs outdoors but symptoms may be exacerbated by indoor exposure. Aspergillus fumigatus is also a common sensitizers. Infections caused by indoor fungi are rare except in severely immunocompromised patients. Aspergillus fumigatus and, less commonly, other Aspergillus species are the most common opportunistic fungi. Cladosporium, Alternaria, and Penicillium infections are extremely rare in normal healthy individuals, and are generally caused by species not common indoors. Virtually all fungi produce toxins depending on genetics and environmental conditions. Almost all exposure to fungal toxins occurs by ingestion. Toxigenic species of Aspergillus are recovered from indoor environments but may not produce their toxins on indoor substrates. Cladosporium produces toxins of apparently low toxicity. Stachybotrys chartarum clearly produces potent toxins, although there remains no good evidence that indoor exposure is sufficient to produce the disease states that are commonly reported.

Conclusions: Overall, allergy exacerbation is by far the most likely response to indoor mold growth, but only in previously sensitized people. The risk of infection and toxicosis related to exposure to these molds is very low. This information should help investigators and physicians to educate their clients about the risk of illness related to mold growth.

CS-112-06 A Case Study on Investigation into Environmental Sources of Legionella Bacteria at a Recreational Spa Facility

M. Canright, J. Martinelli, Forensic Analytical Consulting Services, East Rancho Dominguez, CA

Situation/Problem: In recent years, Legionella bacteria have been the subject of much attention in the scientific community, the public, and the media. As understanding of the bacteria’s ecology, epidemiology, and its role in various water systems has improved, advances have been made in the techniques for the identification and investigation of environmental sources in various settings. The case study will present a risk assessment and investigation into environmental sources during a cluster of Legionellosis at a recreational spa facility. Five cases of Legionnaire’s disease were epidemiologically linked to the spa, which led to investigation by the local regulatory agency. This presentation will review the methods, data collection and findings of the risk assessment investigation and discuss various project considerations and lessons learned.

Resolution: The presentation will detail the investigation process and methodology and review recommended controls that were implemented to reduce the risk of exposure both during and after the investigation conclusion. The role of the local regulatory agencies, the assistance and hindrances associated with their involvement, and involvement of the local media will also be discussed

Results: Several potential environmental sources were identified and addressed during the course of the investigation. Short and long-term administrative and engineering controls were implemented that resulted in successful reductions in sample positivity rate and bacteria concentration and were effective in controlling the onset of new cases linked with the facility. Despite significant improvements, the local regulatory agency enforced additional controls and testing requirements.

Lessons Learned:The case study demonstrates the importance of preparation in maintaining domestic water systems and controlling biofilms and organisms such as Legionella bacteria. The development and implementation of a proactive water management program that includes elements to effectively manage site-specific water related waterborne pathogens risks is essential for maintaining control of water systems and sources.​