Genital infection by Aerococcus viridans in a captive african elephant (Loxodonta africana)

ABSTRACT: Aerococcus viridans is an emerging pathogen for humans and livestock animals, mainly associated with genitourinary infections cases. Its occurrence in wild mammals has never been reported. The aim of this study was to determine the etiological agent associated with clinical a case of a genital infection in a female African elephant (Loxodonta africana). Phylogenetic analysis and antimicrobial susceptibility profile of the isolate were also addressed. The animal presented frequent cases of genital infection with intermittent white secretion. Purulent secretion was sampled and submitted to bacteriological exam. The isolate obtained was thus identified by phenotypic and molecular methods as A. viridans and was found to be similar to human pathogenic isolates in BLASTn and phylogenetic analysis. The isolate was sensitive to almost all antimicrobials evaluated, presenting resistance to ciprofloxacin and norfloxacin. This is the first report of occurrence of A. viridans infection in the genital tract of an African elephant.

Aerococcus viridans is a gram-positive bacterium first described in 1953, being the first characterized species of the genus Aerococcus (WILLIAMS et al., 1953). Later reports recognized the pathogenic potential of the species to humans, causing severe systemic conditions such as meningitis (NATHAVITHARANA et al., 1983), endocarditis (PIEN et al., 1984) osteomyelitis and septic arthritis (TAYLOR et al., 1985).
Recently, this microorganism has emerged as a pathogen of humans and livestock, being mainly associated with acute infections in the genitourinary tract of these hosts (LEITE et al., 2010;TOHNO et al., 2014), as well as related to sepsis in pigs (MARTÍN et al., 2007), subclinical mastitis (LIU et al., 2015) and septicemia in human neonates (accession number Genbank MG576151.1).
Its occurrence in wild mammals has never been reported. It is known that there is a correlation between the maintenance of wild animals in captivity and the increase in the occurrence of diseases due to the continued exposure of these animals to potentially pathogenic microorganisms. Alerting the relevance of the zoonotic character of some of these agents, there are several reports of zoonoses involving wild and domestic animals, as exemplified by the occurrence of Colombo et al. Toxoplasma gondii in captive carnivores (FERREIRA et al., 2018). The pathogenesis of infection by the genus Aerococcus in different hosts is still poorly understood, and fundamental issues such as the normal habitat of pathogenic species and the mode of agent-host interaction are not well known. Recent human cases have shown a change in the virulence profile of this pathogen, in which A. viridans was able to cause serious diseases, even in immunocompetent hosts (YADAV et al., 2018;PARREY et al., 2016). The cases stood out for the acute aspect in the course of the disease. In addition, in one of the cases the transmission of the agent was indicated through the consumption of lobster affected by gaffekia, a disease caused by A. viridans var. homari, suggesting a new transmission route never documented before (YADAV et al., 2018). The aim of this study was to determine the etiology of acute genital tract infection in a female African elephant (Loxodonta africana). Additionally, phylogenetic analyzes and antimicrobial susceptibility profile of the isolate are also addressed.
The elephant had been subjected to surgery in the vestibular region between the vulva and the vagina in the early 1980s. The surgical procedure resulted in a fistula for external communication of the reproductive and urethra. The animal had no signs of dysuria after the surgery. Despite the fistula, the female had two normal gestations and parturitions. Local disinfection was routinely performed with antiseptics and repellent ointments. The animal has been housed at the Belo Horizonte Zoological Garden since 1976, along with another female and an adult male, separated from the females for reproductive control. The animal presented frequent cases of genitourinary infection with intermittent white secretion. During daily management, purulent vulvar secretion was observed. Samples were collected directly from the vaginal canal using a sterile swab and immediately transported to the laboratory for bacteriological examination.
The sample was streaked onto 5% defibrinated sheep's blood and incubated under aerobic and anaerobic conditions at 37 °C for 48 h. Small, non-pigmented, pointy colonies with alpha-hemolytic activity were isolated. The isolate was characterized by Gram staining, catalase and oxidase tests. Identification of the isolate was performed by API 20 Strep ® (Biomerieux, France) according to the manufacturer's recommendations. Bacterial species was confirmed via MALDI-TOF MS analysis performed using a MicroFlex ® LT mass spectrometer (Bruker Daltonics, Bremen, Germany). The parameters for mass range detection and identification score criteria used were those recommended by the manufacturer.
DNA extraction was performed using the Maxwell ® 16 automatic extractor Promega (Madison, Wisconsin, USA) according to the manufacturer's specifications. Extracted DNA was quantified using a NanoDrop ™ spectrophotometer (Thermo Scientific, Wilmington, USA) and stored at -80 °C until further use.
The 16S rRNA gene was amplified by a PCR according to the method described in the literature (FOX et al., 1995). PCR products were purified using the Agencourt ® AMPure ® XP Reagent Beckman Coulter (Brea, California, EUA). Sequencing reactions were conducted using a BigDye™ Terminator Cycle sequencing kit Applied Biosystems (Foster City, California, EUA) and run on an ABI 3500 Genetic Analyzer Life Technologies (Carlsbad, California, EUA).
The 16S rRNA sequence was subjected to BLASTn (http://ncbi.nlm.nih.gov) analysis. Sequence identity >98% was used as the criterion for species identification. The sequence of the isolates was aligned in BioEdit (HALL, 1999) using CLUSTAL W (THOMPSON et al., 1994) with sequences of the strains A. viridans isolates and species of the genus Aerococcus with validly published names and related taxa in GenBank.
The genetic distances matrix was obtained using Kimura's two-parameter model (KIMURA, 1980), and an evolutionary tree was created using the neighbor-joining method (SAITOU, 1987) with Mega7 (KUMAR et al., 2016).
Alpha-hemolytic, gray pointed colonies were obtained in 5% sheep blood agar. The isolate was characterized as gram-positive cocci, negative for catalase and was identified as A. viridans using API 20 Strep.
Identification of A. viridans based solely on biochemical tests is complex as A. viridans has phenotypic and biochemical characteristics that are similar to those of other Aerococcus, leading to it being frequently misidentified as other bacteria when using conventional identification schemes (SENNEBY et al., 2013). To avoid misidentification, the bacterial species was confirmed using MALDI-Tof MS (score > 2.).
This technique has been shown to be a valuable tool for the identification of gram-positive bacteria of clinical importance (CHRISTENSEN et al., 2012). Finally, in BLAST analysis, the isolate had higher than 98% identity of the 16S rRNA gene with A. viridans strains isolated from a case of severe septicemia in human neonates in India (accession number Genbank MG576151.1), strains isolated from genitourinary tract infection in swine in Brazil (accession numbers KR819482.1 and KR819484.1), and from subclinical mastitis in cattle in China (accession number KM096431.1) (Figure 1). This is the first report of Aerococcus viridans associated with a clinical case of infection in a wild animal in a zoo. The veterinarians of the zoo performed a daily cleaning of the fistula with antiseptic products and application of repellent ointments for two weeks. After that treatment, the purulent vulvar secretion was not verified again.
The source of infection of Aerococcus species pathogenic for humans and animals is unknown. It has been isolated from different environments such as air, soil, milk, and invasive infections (CATTOIR et al., 2010;MOHAN et al., 2017;TOHNO et al., 2014). However, its distribution and epidemiology need to be elucidated. PEARCE et al. (2014) compared in a study the composition of the microbiota of women with and without symptoms of urinary incontinence and observed that the presence of bacteria of the genus Aerococcus spp. was more frequent in symptomatic patients. Although the mechanisms of virulence involved in infection are not well known, the presence of preexisting disease is a risk factor for invasion and colonization of the urinary tract by A. viridans (SHANNON et al., 2010). There are no data available regarding the occurrence of Aerococcus spp. in the microbiota of African and other elephant species, and the source of infection in the present case is unknown. The fistula in the genital tract created an external communication between reproductive system and urethra, a condition that probably contributed to A. viridans infection. In the phylogenetic analysis, all strains of the genus Aerococcus clustered in a single branch. The elephant isolate grouped in a common cluster of species of A. viridans, and in a subgroup of pathogenic strains of humans and animals. High homology of the isolate from this study with pathogenic strains in other hosts reinforces the hypothesis of the pathogenic potential of the microorganism as a possible cause of the genitourinary tract infection in the female elephant.
The results of the antimicrobial susceptibility test revealed that the isolate presented resistance to ciprofloxacin and norfloxacin and sensitivity to the other antimicrobials evaluated. These results are consistent with the susceptibility pattern of strains for beta lactam antimicrobials isolated from humans and animals, as well vancomycin, similar to those described by MARTÍN et al. (2007) and MOHAN et al. (2017), although highly resistant strains have been reported in human bacteremic patients (UH et al., 2002). Recently there have been reports of changes in the susceptibility profile of species of the genus Aerococcus for the fluoroquinolones. Resistant strains have emerged in pictures of urinary infections in humans (CATTOIR et al.,2010;MOHAH et al., 2017).
Although the fluorquinolone class antimicrobials tested in this study are not recommended for veterinary use, the data obtained are relevant because they demonstrate the presence of resistance to a group of highest priority critically important antibiotics for humans (WHO, 2016). The phenomenon of bacterial resistance may be associated with the induction of selective pressure due to the prolonged and / or inappropriate use of antimicrobials in human and animal medicine. Greater dissemination and selection of this resistance may compromise the usefulness of a valuable class of antimicrobial agents in public health, reserved for severe cases of infection and also considered a class of choice in the treatment of genitourinary infections in humans (HOOPER, 2002). This is the first report of Aerococcus viridans infection in the genital tract of a female African elephant. The genetic similarity with human and animal strains demonstrates that this emergent pathogen has a broad range of susceptible hosts.