Identification and Differences in Antimicrobial Susceptibility of Lactococcus garvieae from Farmed Grey Mullet ( Mugil cephalus ) and Non-Grey Mullet in Southern Taiwan

Streptococcal infection is a main infectious diseases for farmed grey mullet (Mugil cephalus). This study were to identify spreptococcal species in diseased farmed grey mullet and to investigate differences in susceptibility to 13 antibiotics and in genotypes between the stains from the grey mullet and non-grey mullet. 170 samples from diseased farmed grey mullet were collected from three county in 2013 -2016. Multiplex PCR identified L. garviea (146) as the main pathogen, S. agalactia (9), S. dysgalactiae (19), and double infection (5), but no S. iniae. The prevalence changed annually and differed among three counties. Pulsed-field gel electrophoresis (PFGE) analysis demonstrated identical genotype with an ApaI-digested DNA pattern. Disc diffusion results demonstrated differences in antibiotic susceptibility between the strains from grey mullet (146) and non-grey mullet (30). Almost all strains resisted to clindamycin and all strains were susceptible to six antibiotic in grey mullet and 4 antibiotics in non-grey mullet. The reduced susceptible strains was more in non-grey mullet than grey mullet group. The reduced susceptible strains were observed the highest in 2014 and in Chiayi county and decreased from 2014 to 2016. However, the strains with reduced susceptibility to ceftriaxone, cirpofoxacin, moxifloxacin, tetracycline for human treatment were observed.


Introduction
Grey mullet (Mugil cephalus Linnaeus) is a main farmed fish species with high economic value in Southern Taiwan. These fish are mainly farmed for their mullet roe, which is a type of food. In a cultured environment, diseases are key determinants of whether grey mullet can be harvested. A study investigating diseases in brood grey mullet during the cultivation period revealed that streptococcosis is one of the major bacterial diseases [1]. However, not all of the Gram-positive streptococcus isolated in that study were Streptococcus spp. Therefore, further identification was required [2].
Of course, studies conducted in different years are crucial references for strain infection epidemiology. Streptococcal infection is a threat worldwide. Earlier studies have been unable to identify the strain down to the species level [3,4]. Currently, at least 14 types garvieae (syn. E. seriolicida), L. piscium, L. lactis, and Vagococcus salmoninarum are known to be present in fish [5,6,7,8]. Zlotkin et al. designed L. garvieaea specific primer pairs that could distinguish and identify L. lactis, S. iniae, and Aeromonas salmonicida, and the expected amplification product was 1100 bp [9]. Mata et al. used multiplex polymerase chain reaction (PCR) to analyze streptococcosis, and were able to simultaneously diagnose infections from four different strains, namely S. iniae, S. difficilis, S. parauberis, and L. garvieae [10]. In addition to identifying strains, studies have also adopted technological approaches such as serotyping, biotyping, and pulsedfield gel electrophoresis (PFGE) for molecular typing to study phylogenetics in epidemiology [11,12,13,14.15,16]. Clinically, because Gram-positive streptococcal infection is very common, medicine susceptibility testing for antibiotics treatment is 4 critical for treating fish diseases on site [17,18,19,20]. The public health problem of antibiotic resistance has attracted scholars' attention [21]. The objectives of this study were to establish a method for the rapid diagnosis of Gram-positive streptococcal infection in cultured grey mullet and to clarify the differences between infectious strains by using the results of studies conducted in different years to provide a foundation for subsequent vaccine research. Problems with medical treatment for the main infection strains encountered on site can be identified using the results of antibiotic susceptibility testing conducted in this study. We can also use these results to determine whether fish exhibit resistance to antibiotics not developed for use on fish, which would serve as a warning sign.

Sample collection
A total of 170 samples of the heart, liver, kidneys, spleen, and brain were collected from diseased farmed grey mullet from three counties Yunlin, Chiayi, and Tainan in southern Taiwan from 2013 to 2016. The samples were ground and plated on blood agar (BAP, Difco) containing 5% defibrinated sheep blood. The plates were placed for bacterial growth at 28°C for 24-72 h. This experiment also included 36 L.
garvieae strains from other fish species. Bacterial species were examined by Gramstaining, catalase testing, the Rapid ID 32 STREP system (Bio-Mé rieux Inc, France).
The bacterial strains were preserved in glycerine at −76°C for further use [1,2].

Pulsed field gel electrophoresis (PFGE) analysis
Genotyping of L. garvieae was determined by PFGE analysis with the methods previously [11]. Briefly, overnight bacteria were first embedded in 0.8 % agarose to make the plugs that were treated with lysozyme and then 1 mg/ml proteinase K at 50 °C. After washing with TE buffer, the plugs were digested with the restriction endonuclease ApaI. The macro-DNA fragments were separated by CHEF DRIII (BioRad, Taiwan) using a switching time of 4 s/70 s, 120°, and 6 V for 18 h for the first step and then a switching time of 4 s/70 s, 120°, and 4 V for 6 h. Different genotypes were determined by difference in banding pattern more than three bands. After electrophoresis, the gel was stained with 0.5 mg/mL EtBr for 30 min and the image was recorded under an ultraviolet light illumination. A camera system (Vilber Lourmat, EEC) was used to capture images, which were stored in TIFF format [23]. Laboratory Standards Institute (CLSI) [24]. Streptococcus pneumonia ATCC49619 was used as the reference strain.

Species identification
The multiplex PCR analysis identified L. garvieae, S. agalactiae, and S.

PFGE genotyping of L. garvieae s
Totally 172 L. garvieae were investigated in this study. To differentiate the difference among these strains genetically, we used PFGE analysis and the restriction enzyme ApaI for genotyping. All DNA fragments were smaller than 194 kb, but all strains showed identical patterns (Figure 2), suggesting possibly that single genotype spread in these three counties. Furthermore, all strains lacked plasmid (dada not shown).

Antibiotic susceptibility analysis
Among 13 antibiotics, 146 strains from grey mullet were susceptible to AML, AZM, ERY, DOX, OXT and LVX, but non-susceptible rate differed depending on the antibiotics tested (Table 4) garvieae, and S. dysgalactiae [8,11], indicating the infection of streptococcus species is associated with fish species, maybe due to host immunity and bacterial virulence factors. Previously, L. garvieae commercialized vaccine was applied for farmedctured amberjack and Japanese amberjack for protection, however, the fishes was die by S.
dysgalactiae infection [26]. In this study, occasional double infection occurred mainly in 2014 and in the Yulin and Chiayi counties ( Table 2 and 3). Therefore, multivalent Gram-positive Streptococcus vaccines should be developed in the future because of multiple species infection.
L. garvieae infection is referred to as lactococcosis that has been observed in aquatic animals worldwide [7,27,28]. In Taiwan, L. garvieae has been the primary bacterial parthogen to infect giant freshwater prawns, grey mullet, and trout [29,30,31]. Possibly the grey mullet is more susceptible to L. garvieae infection and resistant to S.
iniae infection. However, the infection may be strain dependent due to differences in the virulence factors they carry. Therefore, PFGE method has been used to distinguish the genotypic differences between strains. The restriction enzyme ApaI digested PFGE analysis could separate 82 L. garvieae strains from Spain and Italy and different animals of fish, dairy cattle, or water buffalo into 19 pulsotypes [16]. Although restriction enzymes may affect PFGE DNA pattern, restriction enzymes ApaI and SmaI for studying L. garvieae genotypes showed identical pulsotype [23]. In our study, the presence of one pulsotype implies that the L. garvieae exists commonly in the aquatic farmed grey mullet in Taiwan [ Figure 2].

Antibiotic susceptibility testing
Antibiotic susceptibility to 13 tested antibiotics differed between the strains from grey mullet and non-grey mullet, such that grey mullet strains were susceptible to 6 antibiotics, including AML, DOX, ERY, LEV, LIN, and OXT; while non-grey mullet strain only susceptible to AZM, ERY, and LEV and showed more reduced susceptible strains, except for antibiotics CLI and MXF (Table 4). These results indicate possible that the antibiotics are used differently for fish species. were found in This study conducted antibiotic susceptibility testing on the primary bacterium L. garvieae in grey mullet. Thirteen types of antibiotics disks were selected. Especially, the highest reduced susceptible strains was located in Chiayi county. Therefore regional antibiotic used shall be controlled more strictly. Antibiotic use can control clinical symptoms in sick fish, while the fishes that become the carrier for these bacterial species makes this disease difficult to eradicate [32].