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Polymicrobial Abscess Following Ovariectomy in a Mouse Victoria E Eaton et al. BMC Veterinary Research (2019) 15:364 https://doi.org/10.1186/s12917-019-2125-0 CASE REPORT Open Access Polymicrobial abscess following ovariectomy in a mouse Victoria E. Eaton1,2, Samuel Pettit1, Andrew Elkinson1, Karen L. Houseknecht1, Tamara E. King1,2 and Meghan May1* Abstract Background: Ovariectomy is a common procedure in laboratory rodents used to create a post-menopausal state. Complications including post-surgical abscess are rarely reported, but merit consideration for the health and safety of experimental animals. Case presentation: A female C57/black6 mouse was ovariectomized as part of a cohort study. At Day 14 post- surgery, she developed a visible swelling on the right side, which 7 days later increased in size over 24 h, leading to euthanasia of the animal. Gross pathology was consistent with abscess. A core of necrotic tissue was present in the uterine horn. Abscess fluid and affected tissue were collected for Gram stain and bacteriological culture. The abscess core and fluid yielded three distinct types of bacterial colonies identified by 16S ribosomal RNA sequencing as Streptococcus acidominimus, Pasteurella caecimuris, and a novel species in the genus Gemella. Conclusions: This is the first report of polymicrobial abscess in a rodent as a complication of ovariectomy, and the first description of a novel Gemella species for which we have proposed the epithet Gemella muriseptica.Thispresentation represents a potential complication of ovariectomy in laboratory animals. Keywords: Ovariectomy, Abscess, Polymicrobial abscess, Mouse, Streptococcus acidominimus, Pasteurella caecimuris, Gemella, Gemella muriseptica Background [7]. Post-surgical infection can be difficult to detect in Ovariectomy (OVX) is a commonly-used model of post- rodents lacking overt sickness behaviors, perhaps leading menopausal age in rodent models of osteoporosis and to an underestimation of its rate of occurrence. Here we bone fragility, impacts of menopause on dopaminergic report a case of polymicrobial abscess following OVX in neurons and other forms of neurodegeration, and adult- a mouse exhibiting normal behavior and no overt signs hood cell differentiation [1–4]. of distress. Infection and specifically abscess is known as a poten- tial complication of survival surgeries in laboratory mice [5]; however, information on the frequency and most Case presentation common causative agents is surprisingly sparse. The History most common agents of spontaneously occurring skin A female C57/black6 mouse (Charles River Laboratories, abscess in pet rodents are Staphylococcus aureus, Wilmington, MA) underwent OVX as previously de- Streptococcus pyogenes,andPasteurella pneumotropica; scribed [8] as part of an 8 animal cohort. All surgical however, (naturally occurring) intra-abdominal abscess tools and silk sutures were sterilized prior to use, and is rarely discussed [6]. As prey animals, mice instinct- the surgical field was disinfected with chlorhexidine ively do not display overt signs of distress during moder- followed by betadine. The surgical site on the mouse ate pain or sickness as a means of avoiding predation was sterilized with 70% ethanol followed by betadine prior to incision. All OVX procedures for the cohort * Correspondence: [email protected] proceeded unremarkably. 1Department of Biomedical Sciences, University of New England, 11 Hills Beach Road, Biddeford, ME 04005, USA Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Eaton et al. BMC Veterinary Research (2019) 15:364 Page 2 of 6 Fig. 1 Post-Surgical Evaluation. The case study subject mouse had no apparent swelling prior to OVX (panel a), and slight swelling became apparent 2 weeks post-OVX (panel b; black arrow). The abscess increased substantially in size overnight during Week 3 (panel c; white arrow) leading to the immediate euthanasia of the animal Clinical findings reagents (Invitrogen via Fisher Scientific, Waltham, MA), Two weeks following OVX, a slight swelling became ap- and the 16S rRNA gene was amplified as previously de- parent on the right side of the abdomen (Fig. 1b). Close scribed using the following primer sequences: 5 AGA monitoring indicated minimal growth of the swelling GTTTGATCCTGGCTCAG 3 ; 5 ACGGCTACCT and no apparent impacts on behavior, weight, or food in- TGTTACGACTT 3 [11]. Amplicons were subjected to take. No other signs of distress such as porphyrin rings Sanger sequencing using standard di-deoxy labelling around the eyes or rough hair coat or hair loss were ob- methods (GeneWiz, Inc. Piscataway, NJ). Bacterial identi- served by the experimenters or care staff. On days 20 to fication was made via BLAST analysis [12]. 21 post-OVX, the swelling increased substantially in size within 24 h (Fig. 1c). Though the subject was still showing Bacteriological findings no signs of distress or sickness behavior, she was eutha- The first colony type could only be cultivated on SP-4 nized because overt swelling/abscess is a humane end- agar and consisted of Gram positive diplococci (Fig. 2b). point criterion for rodents [9]. Euthanasia was carried out Ribosomal RNA sequencing identified this organism as a by pumping 100% CO2 into a sealed chamber at 25% member of the genus Gemella, but the highest identity chamber volume per minute, per approved IACUC proto- (Gemella palaticanis, GenBank accession JN713253) was col. No other animals in the cohort developed abscesses. only 95%. Threshold for ribosomal identity between bac- Postmortem evaluation was consistent with post- terial species is widely considered 97.5% [13]; therefore, surgical abscess. Purulent exudate was apparent into the this isolate likely represents a novel, previously unde- peritoneal cavity, and a small, walled-off abscess core scribed species. The second colony type organisms were was located within the uterine horn. Exudate from the Gram positive cocci that formed long chains (Fig. 2c) peritoneum and the abscess core were subjected to bac- and were identified as Streptococcus acidomini- teriological culture for typical and atypical organisms mus (98% identity with S. acidominimus strain LMG using blood agar (Remel via Thermo Fisher Scientific, 17755, GenBank accession NR_104972). The third col- Waltham, MA) and SP-4 agar supplemented with 10% ony type organisms were Gram negative rods (Fig. 2d) glucose and 15% fetal bovine serum [10]. Both speci- and identified as Pasteurella caecimuris (99% identity mens yielded growth of two colony types on blood agar with P. caecimuris strain AA-424-CC-1, GenBank acces- and three on SP-4 (Fig. 2a). sion NR_144618). Diagnostic procedures Gemella Species Characterization Colonies were propagated in axenic broth culture (SP-4 An unrooted, neighbor-joining tree based on 16S riboso- broth) for preservation, Gram stain, and molecular identi- mal RNA sequence from several Gemella species was fication. Genomic DNA was extracted using EasyDNA generated using MEGA version 7.0 [14](Fig. 3). Input Eaton et al. BMC Veterinary Research (2019) 15:364 Page 3 of 6 Fig. 2 Bacteriological Identification. Three colony types were recovered on SP-4 agar (panel a). Each type was identified as a distinct organism, as shown by Gram stain in panels (b-d). S. acidominimus (c) and P. caecimuris (d) showed classic morphology and arrangements of their respective genera. The novel Gemella species (‘G. muriseptica’) were Gram positive diplococci (Panel b inset, arrow), and bacteria tended to aggregate serum proteins present in the SP-4 broth Fig. 3 Phylogeny of Gemella muriseptica. An unrooted neighbor-joining tree was generated based on 16S rRNA gene sequences from several species in the genus Gemella, as well as uncultivated sequences obtained from mammalian metagenomic studies. G. muriseptica is a divergent member of the genus, and is closest branching relative is Gemella asaccharolytica Eaton et al. BMC Veterinary Research (2019) 15:364 Page 4 of 6 sequences were obtained from GenBank, and included during G. morbillorum monoinfection [15, 16]. Because those from G. palaticanis, Gemella sanguinis, Gemella all three bacterial isolates were cultivated from both parahaemolysans, Gemella haemolysans, Gemella cuni- specimens, it is not possible to determine the order of culi, Gemella morbillorum, Gemella asaccharolytica, infection. Gemella species Oral Strain, and three uncultivated spe- This case features the report of a novel Gemella spe- cies from metagenomics studies (‘uncultivated bacterium’ cies. While no members of the genus have been detected ELU0166, T0751, and J5–76). Phenotypic characteriza- in rodents previously, metagenomic studies show that tions were made using the BBL Crystal Identification re- they are common inhabitants of the oral cavity and in- agents (Becton, Dickinson and Company, Franklin Lakes, testinal tracts of other mammals and can be detected in NJ) (Gram Positive and Gram negative non-fermenter re- wastewater [17–20]. In addition,
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