MATERIALS AND METHODS

Selection of turtle species: Forty one pet turtles of seven commercially popular species were bought from pet shops and online markets in Korea. The turtles were purchased with an average weight of 15±2 g, carapace diameter of 40±5 mm and were under 4 weeks of age. All of the turtles were healthy and no clinical signs of diseases were inspected. There was no antimicrobial therapy applied to the turtles. Among the 41 pet turtles, 2 alligator snapping turtles (Macroclemys temminckii), 2 African sideneck turtles (Pelusios castaneus), 14 Chinese stripe-necked turtles (Ocadia sinensis), 3 peninsula cooters (Pseudemys peninsularis), 10 river cooters (Pseudemys concinna concinna), 3 western painted turtles (Chrysemys picta belli) and 7 yellow-bellied sliders (Trachemys scripta scripta) were used for the study.

Isolation and identification of C. freundii: Fecal samples from the turtles were enriched in tetrathionate broth (MBcell Ltd., Seoul, Korea) by incubation at 37°C for 24 h. Enriched samples were streaked onto MacConkey agar (MBcell Ltd., Seoul, Korea) and incubated at 37°C for 24 h. Pinkish colonies assumed for Citrobacter spp., were subcultured onto xylose lysine deoxycholate agar (MBcell. Ltd, Seoul, Korea) and incubated at 37°C for 24 h. Several biochemical tests such as citrate test, indole test and H₂S test were performed to identify C. freundii. Species status of all bacterial isolates were confirmed by 16S rRNA gene sequencing at Cosmogenetech Co., Ltd. (Daejeon, Korea) using universal primers 518F and 800R.

Antimicrobial susceptibility testing: Susceptibility pattern of 29 C. freundii isolates were detected for nalidixic acid, ciprofloxacin and ofloxacin by disk diffusion test on Mueller-Hinton agar (MBcell Ltd., Seoul, Korea). Minimum inhibitory concentrations (MIC) of nalidixic acid (1-512 mg L^–1), ciprofloxacin (0.125-64 mg L^–1) and ofloxacin (0.125-64 mg L^–1) were determined by broth microdilution method in a 96-well microtiter plate²². All susceptibility testing were conducted according to the recommendations of Performance Standards for Antimicrobial Susceptibility Testing; Clinical and Laboratory Standards Institute²³.

PCR amplification of qnr and aac(6')-Ib genes: The PCR was carried out to detect qnrA, qnrB, qnrS and aac(6')-Ib genes. The primer details were acquired from Cattoir et al.²⁴, Mammeri et al.²⁵ and Chenia²⁶ as shown in Table 1. The PCR mixture 25 μL, contained 12 μL Quick Taq HS DyeMix (Toyobo Co., Ltd., Japan), 9 μL PCR water, 1 μL DNA template and 1 μL of each primer. The thermal cycle for amplification of qnrB gene consisted of 10 min initial denaturation at 94°C, 32 repeated cycles of 45 sec at 94°C, 45 sec at 53°C, 1 min at 72°C and a final extension at 72°C for 10 min. The qnrA and qnrS genes were amplified using conditions; 95°C for 10 min, 35 cycles of 95°C for 1 min, 56°C for 1 min, 72°C for 1 min and 72°C for 10 min. The PCR conditions for amplifying aac(6')-Ib were 94°C for 45 sec, 55°C for 45 sec, 72°C for 45 sec and 72°C for 10 min with 34 repeated cycles. The PCR products were checked in 1.5% (W/V) agarose gel using gel loading buffer with DNA stain (Jena Bioscience GmbH, Germany). The amplicons were purified using Expin^TM PCR SV purification kit (GeneAll, Korea) and sequenced by Cosmogenetech Co., Ltd. (Daejeon, Korea). Acquired aac(6')-Ib sequences were analyzed by comparing with the published sequences in GenBank database.

Detection of mutations in quinolone resistance determining region: The PCR was performed to detect mutations in QRDR of gyrA gene of C. freundii using previously described primers and conditions²⁵ (Table 1). The PCR products of gyrA gene were purified using Expin^TM PCR SV kit (GeneAll, Korea) and sequenced by Cosmogenetech Co., Ltd. (Daejeon, Korea). Acquired sequences were subjected to detection of mutations by comparison with a previously reported gyrA reference sequence²⁷. Analyzing and comparison of QRDR sequences were performed using Mutation Surveyor V5.0.1 (Softgenetics LLC, USA) software.

RESULTS

Isolation of C. freundii from pet turtles: Twenty nine C. freundii strains were isolated from 41 individual turtles as follows: (1) African sideneck turtle, (2) alligator snapping turtles, 11 Chinese stripe-necked turtles, 2 peninsula cooters, 8 river cooters, 2 Western painted turtles and 3 yellow-bellied sliders.

Quinolone resistance patterns in C. freundii isolates: The disk diffusion and MIC results are shown in Table 2. All tested isolates showed nalidixic acid resistance in disk diffusion test. The isolates from river cooters and Chinese stripe-necked turtles showed the distinctive quinolone resistance patterns. Five isolates from river cooters were resistant against all quinolones in disk diffusion test. Except for only three isolates from Chinese stripe-necked turtles which were intermediately resistant to ciprofloxacin, rest of the isolates obtained from the other turtle species showed susceptibility to ciprofloxacin and ofloxacin.

Meanwhile, 4 out of 8 isolates from river cooters exhibited higher MIC values for nalidixic acid, ciprofloxacin and ofloxacin. Bacterial strains isolated from the other turtle species with low MIC value were also noticed. Only four isolates from Chinese stripe-necked turtles showed higher MIC values against ciprofloxacin.

Detection of qnr and aac(6’)-Ib-cr genes: The qnrB gene was found to be the most prevalent among C. freundii isolates. Thirteen from 29 (51.17%) strains were observed to harbor qnrB gene. On the contrary, the qnrS gene was found in seven isolates (24.14%).

Table 1:	Nucleotide sequence of oligonucleotide primers used in the study for detecting quinolone resistance genes

Table 2:	Quinolone resistance patterns of C. freundii isolated from pet turtles and their quinolone resistance gene contents
aC. freundii isolates from African sideneck turtle, Chinese stripe-necked turtle, peninsula cooter, river cooter, Western painted turtle, alligator snapping turtle and yellow-bellied slider were indicated as AF, CSN, PC, RC, SN, WP and YB, respectively. bDisk diffusion zone diameters (mm): NA30: Nalidixic Acid (30 μg), CIP5: Ciprofloxacin (5 μg) and OFX5: Ofloxacin (5 μg), S: Susceptible, I: intermediate, R: Resistant were designated using breakpoints described by the Clinical Laboratory Standards Institute (CLSI)23. The MIC (μg mL–1): S: Susceptible, I Intermediate, R Resistant were designated using breakpoints described by the Clinical Laboratory Standards Institute (CLSI)23 following MIC determination with broth microdilution method22

Only four isolates (13.79%) contained aac(6')-Ib-cr gene. Yet, qnrA gene was not detected in any of the isolates (Table 2). The qnr and aac(6')-Ib-cr genes were frequently encountered in the strains from river cooters.

Mutation determination in QRDR region of gyrA gene: Mutations in QRDR region of gyrA gene was detected in 8 out of 29 (27.59%) isolates (Table 2). Among them, five strains that showed less susceptibility to quinolones in disk diffusion and MIC test were occupied with gyrA alterations resulting in the amino acid substitution of Thr83-Ile. Three isolates having low MIC values to quinolones also had a single mutation in the same amino acid position (Thr83-Ile) of gyrA gene.

DISCUSSION

As an opportunistic pathogen, C. freundii has been found to cause several infections both in humans and animals. In the meantime, it has been known to develop antimicrobial resistance, especially to quinolones. Thus, the study about antimicrobial resistance focusing the resistance of C. freundii against quinolones owns significance.

The current study could characterize C. freundii isolates from pet turtles showing both resistance and susceptibility to quinolones. With regards to qnr gene characterization, the isolates carrying qnrB were found in both resistant and susceptible C. freundii isolates. Similar results were observed in the previous studies^6,12,28. The qnrB was found most frequently in C. freundii than other Enterobacteriaceae. About 75% of qnrB alleles were discovered in the C. freundii isolates and most of them were isolated from human clinical cases²⁹. Furthermore, the qnrS gene was mostly present in the isolates having higher MIC values against quinolones while some other studies also noted that the qnrS positive human isolates were susceptible to nalidixic acid and ciprofloxacin^28,30.

Interestingly, the aac(6’)-Ib-cr gene was found in the isolates that were either less susceptible to quinolones in MIC and disk diffusion test or harboring at least one or two qnr genes. Besides, the strains carrying both qnrS and aac(6')Ib-cr were highly resistant against tested quinolones. On the other hand, the majority of the qnrS and aac(6')-Ib-cr positive strains were isolated from river cooters and Chinese stripe-necked turtles. As the qnrS and aac(6')Ib-cr genes are predominant in the plasmid of these bacterial strains, the possibility of horizontal transfer of these genes between the bacterial flora of turtles and human should not be under valued. Although the presence of qnrA in C. freundii was recorded by Yang et al.³¹, none of the isolates showed qnrA gene in this study. Limitations in the transmission of qnrA between organisms could be the reason as a previous study indicated³⁰. Also, another study revealed that the increasing rate of qnrA gene may not cause the high resistance level of quinolones³².

The mutations in QRDR of gyrA gene were note worthy in current study. The alteration in gyrA is a generic feature of quinolone resistance in bacteria and most of the alterations are located in the first half of gyrA¹⁰. Particularly, the amino acid codons 83 and 87 in gyrA gene displayed typical alterations in clinical isolates¹³. According to Navia et al.³³, C. freundii strains which were having Thr83-Ile alteration exhibited a higher level of resistance to quinolones. It has been identified that the Thr83-Ile mutation arises due to the substitutions at the nucleotide 248th position in gyrA. The study noted that a single alteration in position 83 of gyrA is adequate for establishing a high level of nalidixic acid resistance (MIC>024 μg). Another study indicated that single amino acid alteration in gyrA caused a greater level of ciprofloxacin resistance⁸. The present study could find five C. freundii isolates which were highly resistant to ciprofloxacin (MIC 4-64 μg mL^–1) and nalidixic acid (MIC 64 μg mL^–1) showing Thr83-Ile gyrA mutation.

Nevertheless, a few isolates were susceptible to ciprofloxacin and nalidixic acid in MIC test although they have the mutation in gyrA. The same outcome was encountered in detecting qnr genes in which the possession of qnr genes was not limited to resistant strains. Even though it is controversial, similar results have been reported in C. freundii and also in Aeromonas spp., pointing out the necessity of investigating the organismal internal factors and mechanisms which could suppress or alter the gene expression^25,26.

CONCLUSION

It can be concluded that pet turtle-borne C. freundii strains are a reservoir of chromosomal and plasmid-mediated quinolone resistance determinants. The prevalence of transferable quinolone resistance genes in these bacterial strains play a significant role both in human and anima medicine. Therefore, further studies focusing on characterization of resistance genes related to other antimicrobial groups in C. freundii from pet turtles are highly recommended.

SIGNIFICANCE STATEMENT

The prevalence of quinolone resistance genes in the C. freundii isolates from pet turtles are alarming their prospective pathogenicity leading into public health concerns.

ACKNOWLEDGMENTS

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01060638).