Abstract

Each year animal shelters provide essential care for millions of animals, many of which enter the shelter without receiving any prior veterinary care. Animal shelter housing typically has high occupancy of highly stressed, potentially immunosuppressed animals. Consequently disease transmission is a major concern. To protect both animal and human health, animals are vaccinated and treated with antibiotics; additionally most animal housing units are cleaned daily with disinfectants. The use of antibiotics and disinfectants may promote the spread of resistance traits in resident bacterial populations. Here we propose a study to characterize bacterial and fungal community composition and the presence of antibiotic and disinfectant resistance genes in the microbiota residing on surfaces in an animal shelter.

Objectives

1. We will use amplicon sequencing to characterize the composition of bacterial and fungal communities occurring on surfaces in an animal shelter. By identifying what bacteria and fungi occur here, we can begin to understand sources for these communities (such as animal host, soil, air, water) and identify potential effects that different environmental factors may have on microbial persistence in these building reservoirs (such as use of disinfectants, proximity to windows, moisture, sunlight). These findings can help to inform practices within animal shelters such as the use of ventilation and disinfectants to protect animal health.

2. We will use metagenomic sequencing to identify the presence of genes associated with antimicrobial and antibiotic resistance as well as biofilm formation in these communities. This part of the study provides an important link between describing what microbes are present on building surfaces and identifying what traits might help them to persist in such relatively extreme environments. This study will help in the development of strategies to manage the development of antimicrobial and antibiotic resistance in an intensive housing environment.

Background

Animal shelters house a high density of stressed animals, providing the opportunity for the emergence and transmission of infectious diseases [1]. The US Humane Society estimates that between 6 and 8 million companion animals enter animal shelters in the United States each year. Many of these animals have not received any prior veterinary care or vaccination. Numerous pathogens occur in the animal shelter environment, including beta-hemolytic Streptococci, feline herpesvirus I, canine distemper virus, canine influenza virus, feline calicivirus, parvoviruses and feline infectious peritonitis [1]. Fungal infections such as ringworm are also of major concern [2]. To minimize disease outbreaks in this intensive housing environment, shelter staff use vaccination, antibiotics and disinfectants to protect both animal and human health.

The widespread use of antimicrobial products is accelerating the spread of antibiotic and antimicrobial resistance. Drug resistant bacterial strains have been identified in animal shelters: bacteria with multidrug resistance were isolated from both dog kennels [3] and cat housing areas [4]. Increased biocide resistance may cause increased antibiotic resistance because resistance genes can be located in close proximity and can share common resistance mechanisms (e.g., [5], [6], [7], [8], [9]). Furthermore, antibiotic resistance can be transmitted from animals treated with antibiotics to humans working in close proximity to them [10].

Shelter animals and staff may acquire drug resistant bacterial strains in shelters and carry them into local communities. According to the ASPCA about 2.7 million companion animals are adopted from shelters each year. Many people live in close contact with their pets; studies of dog owners found that they share both gastrointestinal bacteria [11] and skin bacteria [12] with their dogs. Our pets harbor and shed diverse microbial communities that may shape the microbial composition of their human companions [12] through both direct and indirect contact. Although most research has focused on the effects of human occupancy in buildings, animal skin, hair, fecal material, saliva and ectoparasites such as fleas and mites are likely to contribute significantly to the microbial communities associated with indoor surfaces and air [13], [14], [15].

Here we propose a pilot study to characterize the microbial communities occurring on surfaces in an animal shelter. First we will use phylogenetically informative markers to characterize the diversity of bacteria and fungi occurring in these samples. Then we will sequence the metagenome for a subset of these samples to identify genes that may promote persistence in this environment, including those that affect the ability to form biofilms to colonize surfaces, such as amyloid fibers called curli promote adhesion on surfaces and human epithelial cells (found in Enterbacteriaceae), as well as resistance to antimicrobial disinfectants and antibiotics.

Research Plan

We will focus on the holding area for dogs in an open intake municipal animal shelter. The dog holding area contains 52 individual indoor single compartment kennels, 80” long x 40” wide x 80” high. Kennel floors are concrete and walls are ceramic tile covered with concrete block. Kennels are arranged linearly with one central aisle and 2 side aisles. The ventilation system draws air from the outside into the the holding area and exhaust is pulled out by ceiling fans. The kennel also has open windows and doors in summer. Dogs brought into the shelter are subject to a five day holding period and during this period they cannot be walked. Each kennel is cleaned daily to remove animal waste with accelerated hydrogen peroxide sprayed by a pressure washer.

We will collect surface samples from throughout the dog holding area. Surface samples will be collected from the floors, walls and ledges using a 10cm x 10cm grid and sterile, dry cotton swabs (Puritan brand). We collect three samples from each kennel (52 kennels x 3 surfaces = 156 samples). We will record whether a kennel was currently occupied. We will also collect samples from the walkways, drainage system, window ledges and outside each entrance to the dog holding area. Samples will be stored at -80 °C until DNA is extracted.

DNA will be extracted using MoBio PowerSoil kits with an additional lysis step to maximize extraction efficiency for low biomass, swab samples. We will use sterile scissors to cut the cotton head of the swab directly into provided PowerBead tubes containing solution C1. The tubes will be incubated at 65 °C for 10 minutes and vortexed horizontally for 10 minutes. Subsequently, we will follow the standard kit protocol. DNA samples will be stored at -80°C prior to preparing Illumina libraries for sequencing.

DNA isolated from surface samples will be characterized for bacterial diversity based on phylogenetically informative markers in the V4 region of 16S rRNA following the methods of [16]. Fungal diversity will be characterized using the internal transcribed spacer (ITS) region 1 of the nuclear ribosomal coding cistron [17]. Samples will be sequenced in multiplexed Illumina MiSeq lanes (using paired-end 250 base pair sequencing). Sequence data will be entered into the standard Quantitative Insights in Microbial Ecology (QIIME 1.5.0) workflow. Each sample will be characterized for taxonomic composition (number and abundance). For presence/absence analyses, representative operational taxonomic units (OTUs) will be clustered at the >97% identity level and an OTU table will be constructed using QIIME’s pick_otus_through_otu_table.py script. We will compare beta diversity using Fast UniFrac scores [18] visualized with principle coordinates analysis. We will compare the temporal sequence of establishment in each kitten and assess temporal changes in microbiome diversity using various nonparametric multivariate methods, discriminant function analysis and association networks. We will use SourceTracker [19] to identify potential sources of bacteria by comparing bacterial OTUs identified in samples from outside, soil, canine and human.

We will sequence the metagenome for a subset of these samples to detect antimicrobial and antibiotic resistance in bacteria occurring on surfaces in the animal shelter environment. Because we expect to have a low amount of input DNA for each sample, we will use the Illumina Nextera XT library preparation kit. We will select, label with barcodes and pool up to 24 samples for sequencing. We will use PhyloSift [20] to perform phylogeny-driven Bayesian hypothesis tests for the presence of bacteria, fungi, viruses and other organisms occurring in these samples. The BacMet database for antibacterial biocide and metal resistance genes will be used to screen our metagenomic data for resistance genes as well as genes associated with biofilm formation [21].

Team & Resources

Jonathan Eisen, Primary Investigator, is an expert in microbial genomics and an advocate for open science.
Holly Ganz, Project Scientist, is a microbial ecologist, who uses genetic and statistical methods to characterize the diversity of microorganisms that live in the environment and in association with plants and animals. Currently she is leading the Kitten Microbiome Project, a citizen science project on the development of the microbiome in neonatal kittens.
David Coil, Project Scientist. mentors undergraduate research projects in microbial ecology and genomics in the Eisen Lab. Co-investigator for Project MERCURRI, a citizen science project focused on bacteria living in the International Space Station. Also performs public outreach in Microbiology through the microBEnet project, which is focused on communication in the context of studies of microbiology of the built environment.
Katherine Dahlhausen, Graduate Student Researcher, Biophysics Program, studies how bacteria tolerate and respond to DNA damage.
Ruth Lee and Leesa Li, Undergraduate Student Researchers, will provide laboratory assistance.

Available resources: 
1. The UC Davis Genome Center integrates experimental and computational approaches to address key problems at the forefront of genomics. The Center is housed in a new research building with state-of-the-art computational and laboratory facilities. The DNA Technologies Core of the Genome Center provides both high throughput sequencing analysis, including Illumina HiSeq and MiSeq.
 2. Dr. Eisen's laboratory occupies roughly 1000 sq. ft. of wet laboratory space in the Genome and Biomedical Sciences Facility. This laboratory includes all the necessary equipment to perform PCR and qPCR, Qubit (for DNA quantification) and microscopy. Dr. Eisen also has office and cubicle space for computational staff, students and postdoctoral fellows that is directly adjacent to the laboratory. The space can comfortably accommodate eight bench scientists and four computational scientists and can accommodate undergraduate researchers.