The presence of bacteria that is resistant to antibiotics in the marine environment can be used as an indicator of marine pollution
(Marinho et al., 2013; Meena et al., 2015). The types of animals that are affected by bacterial antibiotic resistance include cows, goats, canine, seabirds, sea turtles, and marine mammals
(Banik et al., 2016; Prichula et al., 2016; Swetha et al., 2017; Hristov, 2018; Robaj et al., 2018). Previous researchers have reported the presence of species from the genus
Enterococcus during the rehabilitation of cold-stunned Kemp’s ridley turtles (
Lepidochelys kempii)
(Innis et al., 2014). However, to the best of our knowledge, there have been no previous reports of sea turtles infected with
Enterococcus spp. in Taiwan. The Olive Ridley Turtle (
Lepidochelys olivacea) is one of the five sea turtle species commonly encountered in the coastal waters of Taiwan. This species is listed as endangered by the Forestry Bureau, Council of Agriculture, Executive Yuan, Taiwan and as vulnerable by the International Union for Conservation of Nature
(IUCN, 2016). This study reports on the occurrence of a multiple antibiotic resistant (MAR)
Enterococcus faecalis in the elbow joint of an olive ridley turtle. Specifically, we identified and confirmed the presence of
E.
faecalis usinga biochemical test system, PCR-based methods and physiological tests. The antimicrobial resistance patterns and antibiotic-resistance genes of
E.
faecalis were also analyzed to determine the antimicrobial drug resistance profile of this species. ‘‘Sea turtle rehabilitation at the National Museum of Marine Biology and Aquarium (NMMBA) was also conducted with authorization of the Forestry Bureau, Council of Agriculture, Executive Yuan as permits 105 Forest -1.1-conserv-07(6) and AF No.1051700777. These cover the rehabilitation, medical procedure, collection and processing of samples.”
Case history and observations
An olive ridley turtle (24.48 kg) admitted to a sea turtle rehabilitation facility at the National Museum of Marine Biology and Aquarium. The turtle had a curved carapace length of 56 cm, indicating that it was a sub-adult
(Bhupathy and Karunakaran, 2003). The turtle presented clinical signs, including lack of appetite and bilateral elbow joint swelling. Plasma biochemistry analysis indicated an elevated creatine kinase level (16,005 U/L), lactate dehydrogenase level (16,518 U/L), total protein level (5.9 g/dl), albumin level (2.4 g/dl), uric acid level (1.1 g/dl) and calcium level (10.4 g/dl) when compared with values obtained from healthy wild olive ridley turtles
(Santoro and Meneses, 2007; Espinoza-Romo et al., 2018). Radiographs indicated osteolysis of the humerus, radius and ulna (Fig 1).
Treatment and discussion
The turtle’s elbow was disinfected with alcohol and iodine solution, and aspiration of the joint was performed to collect a sample for culture diagnostic testing
(Greer et al., 2003). The turtle was treated with amoxicillin for 7 days, followed by penicillin G for 2 days and a combination of amikacin and amoxicillin-clavulanic acid
(Innis et al., 2014) for a further 30 days.
An aspirate sample was streaked onto a blood agar plate and incubated at 25°C for 2 days. The purified bacterial isolate that we obtained from the infected sea turtle was labeled AD105039. The strain was routinely grown on tryptic soy agar (TSA; Difco) that included 1.5% NaCl at 25°C for 2 days. Using rapid ID 32 STREP and 16S rDNA analysis, the isolate was identified at the species level as
E.
faecalis. We also employed the
E.
faecalis strain BCRC10789 (obtained from the Bioresource Collection and Research Center; Food Industry Research and Development Institute, Hsinchu, Taiwan) as a reference strain. The 16S rDNA exhibited 100% sequence identity with the reference strain
Enterococcus faecalis (GenBank accession number MF144489 and KX608721). The isolate was confirmed by biochemical testing using the rapid ID 32 Strep system (Biomerieux, Marcy I’ Etiole, France) and 16S rDNA assays, which targeted and amplified a 1524 bp fragment using methods previously described by
Russell et al., (2009). Bacterial cultures from blood agar plates were incubated at 25°C for 18 to 24 h and then used for biochemical testing, which was performed in accordance with the manufacturer’s instructions. After 4 h of incubation, we observed color changes indicative of a positive reaction. API profiles were interpreted using the Rapid ID 32 database to identify bacterial cultures. Chromosomal DNA was extracted from the bacteria strain in accordance with the method proposed by
Tsai et al., (2015). Briefly, 16S rDNA fragments were amplified from genomic DNA with the universal bacterial primers 9Fa (5'-GAGTTTGATCITIGCTCAG-3') and 1513R (5'-TACIGITACCTTGTTACGACTT-3') according to protocols described by
Russell et al., (2009). PCR amplification products were analyzed by gel electrophoresis and subjected to sequencing (Genomics, Taiwan). The resulting sequence was manually aligned with sequences of representative strains retrieved from the GenBank database. The 16S rDNA sequences obtained for
E.
faecalis were compared with available sequences from the GenBank database. Sequence alignment was performed using Clustal W within MEGA4 software with parameters set at default values. The phylogenetic tree with 1000 bootstrap resampling replicates was constructed using the neighbor-joining method using MEGA 4 software
(Harwood et al., 2004). The sequence of the PCR-positive sample obtained in the present study was deposited in Genbank [accession numbers: MH752038]. In antibiotic susceptibility tests, the isolate was spread on Mueller-Hinton agar plates and exposed to antibiotic discs that contained ampicillin (10 μg), penicillin (10 μg), doxycycline (30 μg), enrofloxacin (5 μg), erythro- mycin (15 μg), florfenicol (30 μg), nitrofurantoin (300 μg), neomycin (30 μg), cephalothin (30 μg), oxytetracycline (30 μg), amoxycillin/clavulanic acid (30 μg), gentamicin (10 μg), amikacin (30 μg), piperacillin (100 μg), ciprofloxacin (5 μg), vancomycin (30 μg), sulfamethoxazole and trimethoprim (25 μg), spiramycin (100 μg), chloramphenicol (30 μg), ceftiofur (30 μg), azithromycin (15 μg) and amoxicillin (25 μg). The plate was incubated at 25°C for 18h and inhibition of bacteria by chemotherapeutic agents was evaluated in accordance with the Clinical and Laboratory Standards Institute (CLSI) standards
(CLSI, 2016). The strain was evaluated using PCR to detect the presence of three tetracycline resistance genes
viz.
tet(M),
tet (O) and
tet(S) and three macrolide resistance genes
viz.
erm(B),
mef(A) and
msr(D). The primers used for detection of antimicrobial resistance genes are listed in Table 1. Conditions for amplifying these genes were as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 s, primer annealing 30 s and extension at 72°C for 1 min, with a final elongation step at 72°C for 10 min. The PCR products were resolved by electrophoresis and visualized on a 2% agarose gel
(Nguyen et al., 2017).
The Olive Ridley Turtle received medical treatment for approximately 1.5 months. No clinical improvement was observed, and the turtle eventually died. An
Enterococcus spp. was isolated from bacteriologic cultures of the elbow joint of the turtle, which was identified as
Enterococcus faecalis (Table 2) with high identity (99.2%) by the RAPID ID 32 STREP SYSTEM. However the percent agreements in identification were 79% for
Enterococcus sp. by ID 32 STREP SYSTEM has been reported
(Jackson et al., 2004). For correctly identified, PCR amplification followed by sequencing and sequence comparison of 16S rDNA gene has also allowed differentiation of species of enterococci. This isolate also showed PCR-positive results when employing primers targeting 16S rDNA gene loci for
E.
faecalis. BLAST (Basic Local Alignment Search Tool) was used to comparethe DNA sequence from the isolated strain to reference
E.
faecalis sequences from the GenBank database to confirm that the strain was indeed
E.
faecalis (DNA identity: 100%). Moreover, phylogenetic analysis revealed that the DNA sequence of
E.
faecalis obtained from the infected Olive Ridley Turtle shared significant homology with previously reported sequences (Fig 2). We determined that the
E.
faecalis isolated in this study was resistant to 12 tested antimicrobial agents (Table 3) and possessed the tetracycline resistance gene
tet (M); however, no macrolide resistance genes were found (Table 4).
The occurrence of antibiotic resistant bacteria is considered a global ecological problem. Therefore, the identification of resistant bacteria from marine creatures is an important finding. This is the first study to provide evidence of multiple antibiotic resistant
E.
faecalis associated with osteomyelitis in the Olive Ridley Turtle in Taiwan. Our findings are based on results from a biochemical test system, molecular characterization, phylogenetic analysis and an antibiotic susceptibility examination.
The
E.
faecalis isolate showed a high degree of resistance to the antibiotics tested in this study. This could severely limit the effectiveness of antibiotic therapy in the treatment of sea turtles.
E.
faecalis infections have been increasingly recognized in sea turtles. For example, recent investigations have reported the presence of
E.
faecalis in the fecal samples of wild hawksbill (
Eretmochelys imbricata) and green turtle (
Chelonia mydas) along the southern coast of Brazil
(Prichula et al., 2016). In another previous study,
E.
faecalis were isolated from tissues in a logger head turtle (
Caretta caretta;) stranded along the Tuscany coast of Italy
(Fichi et al., 2016). E.
faecalis were also described in cases of septicemia and osteomyelitis in Kemp’s ridley turtles
(Innis et al., 2014). Additionally,
Meena et al. (2015) showed that
E.
faecalis was a common bacteria found in the seawater environment and
Prichula et al., (2016) revealed that
E.
faecalis was the most commson species isolated from the fecal samples of wild marine species, including sea turtles.
In reptiles, enterococci are known for their strong antimicrobial resistance
(Prichula et al., 2016; Rose et al., 2017). The results of antimicrobial susceptibility tests performed in this study revealed that the
E.
faecalis isolate was resistant to 12 antibiotics (doxycycline, enrofloxacin, erythromycin, neomycin, oxytetracycline, gentamicin, amikacin, ciprofloxacin, spiramycin, chloramphenicol, ceftiofur and azithromycin). This makes the
E.
faecalis isolated in this study a multiple antibiotic resistant (MAR) pathogen
(Magiorakos et al., 2012). In our study, although the
E.
faecalis isolate from this clinical case has been shown to be resistant to amikacin, the combination therapeutic option of amikacin and amoxicillin-clavulanic acid was applied for the treatment after thoroughly consideration. First of all, the combination antibiotic therapy is a suggestive strategy for the treatment of serious bacterial infections
(Caputo et al., 1993; Tamma et al., 2012; Ji et al., 2015), such as the infection of
Enterococcus spp, in human
(Caputo et al., 1993) and veterinary clinical medicine
(Papich et al., 2013; Delis et al., 2018). Second, to consider the synergistic bacterial killing effect through the combination antibiotic therapy,
Delis et al., (2018) reported that the combination of amikacin and amoxicillin-clavulanic acid could be very effective for
E.
coli infection. As a matter of fact, the combination of an aminoglycoside (
e.
g., amikacin) with a beta-lactam antibiotic has been recommended in small animals affected by antimicrobial-resistant
Enterococcus infections
(Papich et al., 2013) and the most common successful combination regimens used in
Enterococcus spp. infection in turtles were amikacin with either amoxicillin-clavulanic acid or ampicillin
(Innis et al., 2014). A retrospective study of cold-stunned Kemp’s ridley turtles (
Lepidochelys kempii) in Massachusetts by
Innis et al., (2014) showed that treating turtles infected with
E.
faecalis can be challenging and indicated a combination treatment of amikacin and amoxicillin-clavulanic acid or ampicillin could be applied. Of importance, amikacin should be used for
E.
faecalis infection in turtles for at least one month
(Innis et al., 2014). Therefore, in our study, after considering the
E.
faecalis isolate was highly susceptible to amoxicillin-clavulanic acid and taking the potential benefit of amikacin with a broad-spectrum of antimicrobial activity as one of the most powerful aminoglycosides used in veterinary medicine
(KuKanich and Coetzee, 2008; Papich et al., 2013), the turtle case with
E.
faecalis infection finally received a combination-based therapy of amoxicillin-clavulanic acid and amikacin for a month.
Enrofloxacin-resistant
E.
faecalis and gentamicin-resistant
E.
faecalis were also reported in previous studies on antibiotic resistance in bacteria isolated during the rehabilitation of Kemp’s ridley turtles
(Innis et al., 2014). The development of multi-drug resistance has increased the pathogenicity of enterococci
(Kuzucu et al., 2005). Therefore, decisions pertaining to the type of antibiotic therapy to use in critical cases of
E.
faecalis infections should be based on empirical testing addressing the antimicrobial activity of the infecting bacterial strain.
In this study, the MAR
E.
faecalis isolate was found to harbour the
tet(M) gene. This is consistent with previous research which reported that the
tet(M) gene was frequently associated with tetracycline resistance in enterococci isolates
(Marinho et al., 2013; Prichula et al., 2016). It is possible that
Enterococcus spp. might act as reservoirs of antimicrobial resistant genes and could therefore spread resistance to other pathogenic bacteria in the marine environment
(Marinho et al., 2013). In conclusion, the detection of MAR
E.
faecalis is an important finding. There is always potential for the transmission of MAR
E.
faecalis and/or its antibiotic resistance genes to sea turtles and other marine wildlife. Further research into the antimicrobial resistance patterns of
E.
faecalis is critically important to the conservation of sea turtles and other endangered marine life in the waters surrounding Taiwan.
