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Antibiotic Resistant Bacteria and Commensal Fungi Are Common 2 and Conserved in the Mosquito Microbiome 3 4 Josephine Hyde1, Courtney Gorham1, Doug E

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Antibiotic Resistant Bacteria and Commensal Fungi Are Common 2 and Conserved in the Mosquito Microbiome 3 4 Josephine Hyde1, Courtney Gorham1, Doug E bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Antibiotic resistant bacteria and commensal fungi are common 2 and conserved in the mosquito microbiome 3 4 Josephine Hyde1, Courtney Gorham1, Doug E. Brackney1,2, Blaire Steven1* 5 6 1Department of Environmental Sciences, Connecticut Agricultural Experiment Station, 7 New Haven, CT, USA 8 9 2Center for Vector Biology and Zoonotic Diseases, Connecticut Agricultural Experiment 10 Station, New Haven, CT, USA 11 12 13 14 *Corresponding author 15 E-mail: [email protected] 16 17 18 19 20 21 22 23 24 25 26 Short title: Antibiotic resistant bacteria in mosquitoes 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 1 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 46 Abstract 47 The emerging and increasing prevalence of bacterial antibiotic resistance is a 48 significant public health challenge. To begin to tackle this problem, it will be critical to 49 not only understand the origins of this resistance but also document environmental 50 reservoirs of antibiotic resistance. In this study we investigated the possibility that both 51 colony and field caught mosquitoes could harbor antibiotic resistant bacteria. 52 Specifically, we characterized the antibiotic resistant bacterial populations from colony- 53 reared Aedes aegypti larvae and adults and two field caught mosquito species 54 Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial 55 populations were dominated by isolates belonging to the class Gammaproteobacteria. 56 Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, 57 kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three 58 antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 59 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and 60 clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 61 antibiotic resistant OTUs were identified, although members of an OTU did not always 62 share the same resistance profile. This suggests the clustering was either not sensitive 63 enough to distinguish different bacteria taxa or different antibiotic resistant sub- 64 populations exist within an OTU. Finally, the antibiotic selection opened up a niche to 65 culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) 66 were identified. Two fungal OTUs both classified to the class Microbotryomycetes were 67 commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate 68 that antibiotic resistant bacteria and certain fungi are common and conserved mosquito 2 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 microbiome members. These observations highlight the potential of invertebrates to serve 70 as vehicles for the spread of antibiotic resistance throughout the environment. 71 3 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 72 Introduction 73 Mosquitoes, like many complex organisms, live in association with a microbiome 74 made up of bacteria, fungi and viruses [e.g. 1–5]. Descriptive studies of the mosquito 75 microbiome suggest that the bacterial species richness of the microbiome is relatively 76 low, generally made up of 10 to 50 species [1,6]. As mosquitoes are vectors of blood 77 borne arboviruses, such as West Nile virus, dengue virus, and chikungunya virus, the 78 microbiome has received substantial research as a possible factor that could interact with 79 mosquito vector competence [7,8]. Yet, these studies are hampered by an inability to 80 systematically manipulate the microbiome. 81 The generation of axenic mosquitoes, allowing for establishing gnotobiotic hosts 82 with a controlled microbiome, has only been reported recently [9,10]. Previous studies 83 had relied on antibiotic treatment of the mosquitoes to reduce or “remove” the 84 microbiome prior to experiments [11–14]. However, studies have documented that the 85 mosquito microbiome can harbor antibiotic resistant populations [15,16] and thus 86 antibiotic treatment is more likely to induce a dysbiosis in the microbiome rather than an 87 elimination of the mosquitoes’ microbiota [12]. Furthermore, antibiotic treatment can 88 affect the host’s physiology by inducing mitochondrial dysfunction and oxidative damage 89 [17,18] . In this regard, it is often difficult to distinguish the effects of reducing the 90 microbiome load via antibiotics versus potential effects of the antibiotics themselves. 91 In this study we set out to test how common antibiotic resistant bacteria are 92 among colony-reared and field-caught mosquitoes. We hypothesized that the mosquito 93 microbiome would naturally harbor antibiotic resistant bacterial populations. We further 94 predicted that the wild-caught mosquitoes would harbor larger and more diverse 4 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 95 antibiotic populations due to their increased probability of encountering antibiotics in 96 their environment, as the colony mosquitoes had no history of antibiotic treatment. 97 98 Materials and Methods 99 Rearing and collection of colony mosquitoes 100 Colony reared mosquitoes (Aedes aegypti; Orlando strain) were maintained using 101 standard conditions [9]. Larvae from the same egg hatch were collected from rearing pans 102 with a Pasteur pipette and serially rinsed through three Petri dishes containing sterile 103 water to remove external adhering bacteria. Adult female mosquitoes were collected 24 104 hours post blood feeding (hpbf) from females with a still visible blood bolus to ensure 105 that the mosquito had blood fed, which insured the mosquitoes were in a similar 106 physiological state. 107 108 Testing colony-reared mosquitoes for antibiotic resistance genes 109 To investigate the presence of antibiotic resistance genes in the microbiome of 110 colony-reared mosquitoes we employed a quantitative PCR array. Total DNA was 111 extracted from a pool of three larvae and from the dissected midguts of three female 112 adults 24 hpbf. DNA was extracted with the DNeasy PowerSoil kit (Qiagen) using 113 standard protocols as previously described for mosquitoes [9]. The DNA was employed 114 as a template for a Qiagen 96-well Microbial DNA qPCR Array (Antibiotic Resistant 115 genes). This array detects 84 antibiotic resistant genes across multiple antibiotic classes 116 and also includes two pan bacteria and one positive PCR controls to test for the presence 117 of inhibitors and PCR efficiency. A full list of the genes detected in the assay can be 5 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 118 found on the manufacture’s website. Quantitative PCR was performed on a BioRad 119 C1000 Touch Thermocycler with CFX96 Real-Time System. Thermalcycling conditions 120 consisted of 10 minutes at 95°C, followed by 40 cycles of: 15 seconds at 95°C and 2 121 minutes 60°C and a 1°C/s ramp rate (as recommended by the assay manufacture) with 122 FAM fluorophore detection. Values for cycle quantification value (Cq) were recorded for 123 each well and positive detections were based on the manufacture’s recommendations. 124 125 Field collection of mosquitoes 126 Mosquito trapping was conducted as described previously [19]. Briefly, CO2 (dry 127 ice) baited CDC miniature light traps equipped with aluminum domes were collected 128 from six sites around Connecticut (Fig. 1). Traps were placed in the field in the afternoon, 129 operated overnight, and retrieved the following morning. Adult mosquitoes were 130 transported alive to the laboratory each morning in an ice chest lined with cool packs. 131 Mosquitoes were immobilized with dry ice and transferred to chill tables where they were 132 identified to species with the aid of a stereo microscope (90X) based on morphological 133 characters. Individual females of two species, Coquillettidia perturbans and Ochlerotatus 134 canadensis were identified and separated with sterile tweezers and processed for bacterial 135 isolation immediately after collection from the field. 136 137 Bacterial culturing and isolation. 138 In initial experiments we tested the bacterial recovery from individual colony- 139 reared and field-caught mosquitoes. For the colony mosquitoes an individual was 140 sufficient to recover c.a. 1 x 107 colony-forming units (CFUs; for larvae and post blood 6 bioRxiv preprint doi: https://doi.org/10.1101/670802; this version posted June 13, 2019.
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