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First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa
Veterinary Microbiology 138 (2009) 140–144
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa Zhen-Yu Xie a,b, Yong-Can Zhou a,b,*, Shi-Feng Wang a,b, Bing Mei b, Xian-Dong Xu b, Wan-Yao Wen b, Yong-Qin Feng a,b a b
Key Laboratory of Tropic Biological Resources, MOE, Hainan University, Haikou 570228, PR China Key Laboratory of Tropical Aquatic Biotechnology of Hainan Province, Hainan University, Haikou 570228, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2008 Received in revised form 13 February 2009 Accepted 17 February 2009
Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents. The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Elizabethkingia meningoseptica Pathogenicity Rana tigerina rugulosa Cataract disease
The tiger frog, Rana tigerina rugulosa, was widely distributed in Hainan and other places in South China, but its wild stock has decreased rapidly in the recent 20 years. Because tiger frog was highly efficient in pest control and has a high commercial value, it has been farmed since 10 years in China. Now, frog farming is a fast-growing industry in Hainan, China. However, as frog culture
* Corresponding author at: College of Marine Science, Hainan University, 58 Renming Road, Haikou, Hainan Province 570228, PR China. Tel.: +86 898 66289553; fax: +86 898 66279215. E-mail addresses: [email protected], [email protected] (Y.-C. Zhou). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.02.011
developed, more and more diseases appeared and seriously hampered tiger frog culture. Little information is available about the diseases of tiger frog, except for the virulence properties of motile aeromonads isolated from septicaemic farmed frogs (Pearson et al., 2000). Recently, a very serious cataract disease broke out in Hainan frog farms. It spreads very quickly and has become the most prevalent disease in farmed tiger frog in Hainan. Its clinical signs were ascites, torticollis and opaque eye lens. The diseased frogs were lethargic and most of them died in a few days. Elizabethkingia meningoseptica was frequently isolated from these frogs with cataract disease in the study. E. meningoseptica (previously Flavobacterium meningosepticum, Chryseobacterium meningosepticum; Kim et al.,
Z.-Y. Xie et al. / Veterinary Microbiology 138 (2009) 140–144
2005) is a Gram-negative bacterium widely distributed in nature. It has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings, giving rise to meningitis, pneumonia, septic arthritis, endocarditis and conjunctivitis (Bernardet et al., 2006). E. meningoseptica was also occasionally isolated from diseased turtles, frogs and fish (Vancanneyt et al., 1994; Green et al., 1999; Mauel et al., 2002; Bernardet et al., 2005). In this study, we first determined that E. meningoseptica was the causative agent of this cataract disease in tiger frog and demonstrated why it was difficult to control this disease by chemotherapeutic agents. 1. Materials and methods 1.1. Bacterial isolation In the summer of 2007, six moribund tiger frogs weighing approximately 150 g from three separate farms in Qiongshan County and Wenchang City, Hainan, China, were selected for bacteria isolation. Nearly 70% of the frogs in the sampled farms displayed signs of cataract disease and 80–100% of the diseased frogs died. The frogs were first sanitised with 70% alcohol and dissected in the laboratory. Using a swab, the samples, isolated from the eyes, brains, livers and ascites (abdominal swelling), were all inoculated onto trypticase soy agar (TSA, bioMe´rieux sa, Marcy— I´E´toile, France) at 28 8C for 24 h. The dominant isolates were then purified by streaking and re-streaking on the same agar plates. Pure stock isolates were stored at 80 8C in sterile balanced salt solution (BSS: 0.8% NaCl, 0.11% K2SO4, 0.135% NaH2PO4, 0.005% NaHCO3; pH 7.2) supplemented with 15% glycerol. 1.2. Bacterial identification Strains W0701, W0702 and W0703 were incubated at 28 8C for 24 h in nutrient broth (Oxoid, England) and DNA was extracted following standard procedures. Polymerase chain reaction (PCR) assay was performed in a final volume of 50 ml using a PTC-100TM programmable thermal controller (MJ Research, Inc., England). The reaction mixtures consisted of 5 ml of a 10 PCR reaction buffer (Ex TaqTM Buffer, Takara, Japan), 200 mmol l1 of each of the four deoxynucleotide triphosphates (dNTPs), 1.5 mmol l1 MgCl2, 100 nmol l1 of each primer, 1 ml extracted DNA, 1.25 U Ex TaqTM polymerase (Takara, Japan) and sterile UltraPure water (Sartorius, Germany). The 16S rRNA gene was amplified with the Unip1 (50 -AGAGTTTGATCMTGGCTCAG-30 , M = A/C/G, positions 8–27 in Escherichia coli) forward and Unip2 (50 -GGTTACCTTGTTACGACTT-30 , positions 1492–1510 in E. coli) reverse primers. PCR was performed with the annealing temperature at 58 8C for 2 min. The amplified products were sequenced automatically using an ABI Prism 3730 XL DNA Analyzer System (Applied Biosystems, USA). Strains W0701–W0706 were identified using Gram reaction (KOH method), cellular morphology and colony characteristics on nutrient agar and TCBS agar. Classical phenotypic tests were performed using standard methods according to Holt et al. (1999).
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1.3. Sensitivity of strain W0702 to various chemotherapeutic agents Strain W0702 was tested for sensitivity to 20 chemotherapeutic agents by the disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2002). Briefly, 5 ml of trypticase soy broth (TSB, bioMe´rieux sa, Craponne, France) was inoculated with one loop of culture. The suspension obtained was uniformly spread onto the surface of dry Mueller–Hinton agar (MH, Oxoid, UK) plates using broth-impregnated swabs. The inoculum concentration was approximately 1 108 CFU ml1. The following discs were used: ampicillin (10 mg), streptomycin (10 mg), erythromycin (15 mg), chloramphenicol (30 mg), tetracycline (30 mg), clindamycin (2 mg), kanamycin (30 mg), gentamicin (10 mg), vancomycin (30 mg), azitromycin (15 mg), cefuroxime (30 mg), ceftazidime (30 mg), cefoperazone (75 mg), norfloxacin (10 mg), ciprofloxacin (5 mg), nitrofurantoin (300 mg), furazolidone (300 mg), aztreonam (30 mg), ofloxacin (5 mg) and sulfafurazole (300 mg). The plates were incubated at 28 8C for 18 h and the inhibition of the bacteria by the chemotherapeutic agents was scored. 1.4. Virulence tests of W0702 to tiger frogs Healthy tiger frogs weighing 100 5 g each were held in 100-l tanks supplied with wet pledget at 25–28 8C. Five passages of strain W0702 on frog were performed prior to the virulence tests. The virulence tests with batches of 10 frogs were conducted by intramuscular (IM) injection of bacterial suspensions (1 107 CFU per frog after a 24-h culture) into the leg muscle. Sterile BSS was injected into another group of animals as parallel controls. Each group was duplicated and mortalities were recorded daily for 2 weeks post injection. Reisolation on TSA plates and identification of the bacteria from the brain and the ascites of moribund frogs were performed after bacterial challenge. All animal challenges were carried out following IACUC-approved protocols of Hainan University. 2. Results 2.1. Bacterial isolation Several tens of colonies grew on each plate in all samples after incubation for 24 h on TSA. However, only one type of dominant, white, round colonies and 0.1–0.2 cm in diameter was observed on each plate. Four colonies isolated from each sample were inoculated into TSB for stock culture. All these strains were numbered from W0701 to W0724 (four colonies six frogs). Then the strains W0701, W0702 and W0703 (isolated from the brains of three different frogs) and strains W0704, W0705 and W0706 (isolated from three other frogs) were selected for the following experiment, because the common clinical signs were lethargy, torticolis (presumed brain damage) and cataract (eye damage). 2.2. Bacterial identification The nearly complete 16S rRNA gene sequences of strains W0701 (EU128744), W0702 (EU128742) and
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Table 1 Main phenotypic traits of strains W0701–W0706 and E. meningoseptica. Test methods
Characteristics
Strains W0701–W0706
E. meningoseptica
Microscope
Gram stain Shape
Straight rod
Rods with parallel sides and rounds ends
Growth at
4 8C 25 8C 35 8C 37 8C 40 8C 42 8C
+ + +
NA NA NA + NA
Color
+ Green
NA NA
Color Colony morphology Swarm
+ White 0.1–0.2 cm/round/shiny/raised
+ NA NA
Color Colony morphology
da White 0.1–0.2 cm/round/shiny/raised
db White to yellowb 0.1–0.2 cm/round/shiny
+
db b
Growth on TCBS
Growth on TSA
Growth on McConkey agar
Citrate utilization Malonate utilization
Reactions
Reduction of
NO3 NO2
d
Hydrolysis of
Starch ONPGb Esculin
+ +
+ +
Production of
Indole H2S Gelatinase Urease Oxidase Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolase
+ + + +
d b + d + NA NA NA
Acid production from
Cellobiose Lactose Glucose Sucrose Raffinose Arabinose Rhamnose Trehalose Xylose Fructose Maltose Galactose Mannitol Adonitol Salicin Inositol Aescinate glycoside Sorbitol
+ +
d d d +b +b NA d NA NA NA
ONPG hydrolysis is represent for b-galactosidase production. NA, No data available. a Variable reactions are positive for W0701, W0703 and W0704. b Data from Kim et al. (2005).
W0703 (EU128743) were obtained. They showed 98.6– 98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL reported in Genbank.
All investigated biochemical reactions are shown in Table 1. Briefly, W0701–W0706 were Gram-negative, straight rods, non-motile, positive for oxidase, gelatinase, urease and indole, negative for lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase and H2S.
Z.-Y. Xie et al. / Veterinary Microbiology 138 (2009) 140–144 Table 2 Sensitivity of strain W0702 to various chemotherapeutic agents. Chemotherapeutic agents
Disc content (mg)
Sensitivitya
Ampicillin Streptomycin Erythromycin Chloramphenicol Tetracycline Clindamycin Kanamycin Gentamicin Vancomycin Azitromycin Cefuroxime Ceftazidime Cefoperazone Norfloxacin Ciprofloxacin Nitrofurantoin Furazolidone Aztreonam Ofloxacin Sulfafurazole
10 10 15 30 30 2 30 10 30 15 30 30 75 10 5 300 300 30 5 300
R R R R R R R R S R R R MS R R R R R R R
a
R: Resistant; S: sensitive; MS: moderately sensitive.
They could hydrolyse ONPG and esculin but not starch. Nitrate and nitrite were not reduced as electron acceptors and citrate was not used as a carbon source. Among the 14 saccharides tested, acid was produced aerobically only from glucose and aescinate glycoside. Green colonies were produced on thiosulfate citrate bile salt sucrose agar (TCBS, Difco, USA). Growth on McConkey agar (Difco, USA) was strain depended: only strains W0701, W0703 and W0704 could grow on this medium. No growth occurred at 4 or 40 8C after 2 weeks. All biochemical characteristics tested on strains W0701–W0706 investigated above were consistent with those of E. meningoseptica strains (Holt et al., 1999; Kim et al., 2005) except that acid was not produced from fructose and maltose and that malonate was not used. On the basis of these results, strains W0701–W0706 which are representatives of all the 24 isolates were identified as E. meningoseptica strains. 2.3. Sensitivity of strain W0702 to various chemotherapeutic agents The results of the sensitivity tests of strain W0702 to 20 different chemotherapeutic agents are shown in Table 2. The organism was only susceptible to vancomycin and moderately susceptible to cefoperazone. 2.4. Virulence of strain W0702 to tiger frogs Bacterial cells of strain W0702 were injected into tiger frog and the results showed that W0702 was lethal to tiger frogs. The mortality rate of the infected tiger frog was 80%. Three days following injection with strain W0702, most tiger frogs exhibited the same clinical signs (opaque lens, ascites, torticollis and lethargy) as those observed in naturally infected tiger frogs. Strain W0702 could be reisolated as pure colonies from the brains of all injected frogs and the re-isolated strain was also strongly virulent to healthy tiger frogs.
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3. Discussion All the six isolates (strains W0701–W0706, representatives of the 24 similar strains) obtained from the diseased frogs with typical clinical signs of cataract disease were identified as E. meningoseptica according to their 16S rRNA gene sequences and biochemical characteristics. The few phenotypic discrepancies (shown in Table 1) between the isolates and E. meningoseptica strains were strain dependent. The 16S rRNA gene sequences of strains W0701, W0702, W0703 showed the highest similarity with that of E. meningoseptica strain NTU 870424-IL (accession number is AY468477) whose 16S rRNA gene showed 98.5% similarity with that of E. meningoseptica ATCC 13253. Interestingly, this strain had been isolated from the internal organs of a cultured bullfrog (Rana catesbeiana) with haemorrhagic septicaemia in Taiwan in 1986 (Bernardet et al., 2005). E. meningoseptica was previously reported as the aetiological agent of disease in South African clawed frogs (Xenopus laevis) and leopard frogs (Rana pipiens) (Taylor et al., 1993; Green et al., 1999). The virulence test showed that W0702 was strongly virulent to tiger frog. This is the first report of E. meningoseptica as a pathogen for tiger frog. Certainly, the concurrent infection with this bacterium and virus was possible because no virus test was performed in the study. The strains W0701, W0702 and W0703 were isolated from the brains of diseased frogs, suggesting that E. meningoseptica could break through blood–brain barrier and damage the nervous system. Therefore, the common symptom of diseased frog was torticollis. Neurological signs were considered secondary lesions in bullfrog infected by E. meningoseptica (Mauel et al., 2002). However, they seemed to be primary in tiger frogs as the first clinical sign was torticollis. Although no human case has been reported so far among the staff of frog farms in Hainan, E. meningoseptica is mostly pathogenic for premature or newborn infants and for immunocompromised adults (Bernardet et al., 2006). Since we have frequently isolated E. meningoseptica from the water of frog ponds in Hainan in recent years, it seems that appropriate aquacultural management is essential to avoid this constant threat to frog farming and/or to the staff of frog farms. Antibiograms showed that strain W0702 was resistant to all antibiotics tested except to vancomycin and cefoperazone, which meant that this frog’s pathogen had obtained many antibiotics genes in the long-term evolution process. This explains why this epidemic disease of tiger frog in Hainan was very difficult to control by antibiotic treatment. Hence, a specific vaccine is desirable to control the frog’s disease caused by E. meningoseptica. Acknowledgements This work was supported by Program for New Century Excellent Talents in University, no. NCET-05-0755; grants from the National Natural Science Foundations of China, no. 30660144 and no. 30760190; the Key Project for International Cooperation, no. 2005DFA30610 and the Natural Science Foundations of Hainan Province, no. 80617.
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References Bernardet, J.F., Bruun, B., Hugo, C., 2006. The genera Chryseobacterium and Elizabethkingia. In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H., Stackebrandt, E. (Eds.), The Prokaryotes, a Handbook on the Biology of Bacteria. third ed. Springer, New York, pp. 638–676. Bernardet, J.F., Vancanneyt, M., Matte-Tailliez, O., Grisez, L., Tailliez, P., Bizet, C., Nowakowski, M., Kerouault, B., Swings, J., 2005. Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals. Syst. Appl. Microbiol. 28, 640–660. Clinical and Laboratory Standards Institute, 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, Approved Standard, second ed. M31-A2. Wayne, PA. Green, S.L., Bouley, D.M., Tolwani, R.J., Waggie, K.S., Lifland, B.D., Otto, G.M., Ferrell, J.E., 1999. Identification and management of an outbreak of Flavobacterium meningosepticum infection in a colony of South African clawed frogs (Xenopus laevis). J. Am. Vet. Med. Assoc. 214, 1833–1838.
Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1999. Bergey’S Manual of Determinative Bacteriology, 9th ed. Lippincott Williams and Wilkins, Philadelphia, pp. 83–140. Kim, K.K., Kim, M.K., Lim, J.H., Park, H.Y., Lee, S.T., 2005. Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen. nov. as Elizabethkingia meningoseptica comb. nov. and Elizabethkingia miricola comb. nov. Int. J. Syst. Evol. Microbiol. 55, 1287–1293. Mauel, M.J., Miller, D.L., Frazier, K.S., Hines 2nd, M.H., 2002. Bacterial pathogens isolated from cultured bullfrogs (Rana catesbeiana). J. Vet. Diagn. Invest. 14, 431–433. Pearson, M.D., Hirono, I., Aoki, T., Miranda, R., Inglis, V., 2000. Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa. Dis. Aquat. Organ. 40, 185–193. Taylor, F.R., Simmonds, R.C., Loeffler, D.G., 1993. Isolation of Flavobacterium meningosepticum in a colony of leopard frogs (Rana pipiens). Lab. Anim. Sci. 43, 105. Vancanneyt, M., Segers, P., Hauben, L., Hommez, J., Devriese, L.A., Hoste, B., Vandamme, P., Kersters, K., 1994. Flavobacterium meningosepticum, a pathogen in birds. Int. J. Syst. Evol. Microbiol. 32, 2398–2403.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa Zhen-Yu Xie a,b, Yong-Can Zhou a,b,*, Shi-Feng Wang a,b, Bing Mei b, Xian-Dong Xu b, Wan-Yao Wen b, Yong-Qin Feng a,b a b
Key Laboratory of Tropic Biological Resources, MOE, Hainan University, Haikou 570228, PR China Key Laboratory of Tropical Aquatic Biotechnology of Hainan Province, Hainan University, Haikou 570228, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2008 Received in revised form 13 February 2009 Accepted 17 February 2009
Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents. The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Elizabethkingia meningoseptica Pathogenicity Rana tigerina rugulosa Cataract disease
The tiger frog, Rana tigerina rugulosa, was widely distributed in Hainan and other places in South China, but its wild stock has decreased rapidly in the recent 20 years. Because tiger frog was highly efficient in pest control and has a high commercial value, it has been farmed since 10 years in China. Now, frog farming is a fast-growing industry in Hainan, China. However, as frog culture
* Corresponding author at: College of Marine Science, Hainan University, 58 Renming Road, Haikou, Hainan Province 570228, PR China. Tel.: +86 898 66289553; fax: +86 898 66279215. E-mail addresses: [email protected], [email protected] (Y.-C. Zhou). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.02.011
developed, more and more diseases appeared and seriously hampered tiger frog culture. Little information is available about the diseases of tiger frog, except for the virulence properties of motile aeromonads isolated from septicaemic farmed frogs (Pearson et al., 2000). Recently, a very serious cataract disease broke out in Hainan frog farms. It spreads very quickly and has become the most prevalent disease in farmed tiger frog in Hainan. Its clinical signs were ascites, torticollis and opaque eye lens. The diseased frogs were lethargic and most of them died in a few days. Elizabethkingia meningoseptica was frequently isolated from these frogs with cataract disease in the study. E. meningoseptica (previously Flavobacterium meningosepticum, Chryseobacterium meningosepticum; Kim et al.,
Z.-Y. Xie et al. / Veterinary Microbiology 138 (2009) 140–144
2005) is a Gram-negative bacterium widely distributed in nature. It has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings, giving rise to meningitis, pneumonia, septic arthritis, endocarditis and conjunctivitis (Bernardet et al., 2006). E. meningoseptica was also occasionally isolated from diseased turtles, frogs and fish (Vancanneyt et al., 1994; Green et al., 1999; Mauel et al., 2002; Bernardet et al., 2005). In this study, we first determined that E. meningoseptica was the causative agent of this cataract disease in tiger frog and demonstrated why it was difficult to control this disease by chemotherapeutic agents. 1. Materials and methods 1.1. Bacterial isolation In the summer of 2007, six moribund tiger frogs weighing approximately 150 g from three separate farms in Qiongshan County and Wenchang City, Hainan, China, were selected for bacteria isolation. Nearly 70% of the frogs in the sampled farms displayed signs of cataract disease and 80–100% of the diseased frogs died. The frogs were first sanitised with 70% alcohol and dissected in the laboratory. Using a swab, the samples, isolated from the eyes, brains, livers and ascites (abdominal swelling), were all inoculated onto trypticase soy agar (TSA, bioMe´rieux sa, Marcy— I´E´toile, France) at 28 8C for 24 h. The dominant isolates were then purified by streaking and re-streaking on the same agar plates. Pure stock isolates were stored at 80 8C in sterile balanced salt solution (BSS: 0.8% NaCl, 0.11% K2SO4, 0.135% NaH2PO4, 0.005% NaHCO3; pH 7.2) supplemented with 15% glycerol. 1.2. Bacterial identification Strains W0701, W0702 and W0703 were incubated at 28 8C for 24 h in nutrient broth (Oxoid, England) and DNA was extracted following standard procedures. Polymerase chain reaction (PCR) assay was performed in a final volume of 50 ml using a PTC-100TM programmable thermal controller (MJ Research, Inc., England). The reaction mixtures consisted of 5 ml of a 10 PCR reaction buffer (Ex TaqTM Buffer, Takara, Japan), 200 mmol l1 of each of the four deoxynucleotide triphosphates (dNTPs), 1.5 mmol l1 MgCl2, 100 nmol l1 of each primer, 1 ml extracted DNA, 1.25 U Ex TaqTM polymerase (Takara, Japan) and sterile UltraPure water (Sartorius, Germany). The 16S rRNA gene was amplified with the Unip1 (50 -AGAGTTTGATCMTGGCTCAG-30 , M = A/C/G, positions 8–27 in Escherichia coli) forward and Unip2 (50 -GGTTACCTTGTTACGACTT-30 , positions 1492–1510 in E. coli) reverse primers. PCR was performed with the annealing temperature at 58 8C for 2 min. The amplified products were sequenced automatically using an ABI Prism 3730 XL DNA Analyzer System (Applied Biosystems, USA). Strains W0701–W0706 were identified using Gram reaction (KOH method), cellular morphology and colony characteristics on nutrient agar and TCBS agar. Classical phenotypic tests were performed using standard methods according to Holt et al. (1999).
141
1.3. Sensitivity of strain W0702 to various chemotherapeutic agents Strain W0702 was tested for sensitivity to 20 chemotherapeutic agents by the disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2002). Briefly, 5 ml of trypticase soy broth (TSB, bioMe´rieux sa, Craponne, France) was inoculated with one loop of culture. The suspension obtained was uniformly spread onto the surface of dry Mueller–Hinton agar (MH, Oxoid, UK) plates using broth-impregnated swabs. The inoculum concentration was approximately 1 108 CFU ml1. The following discs were used: ampicillin (10 mg), streptomycin (10 mg), erythromycin (15 mg), chloramphenicol (30 mg), tetracycline (30 mg), clindamycin (2 mg), kanamycin (30 mg), gentamicin (10 mg), vancomycin (30 mg), azitromycin (15 mg), cefuroxime (30 mg), ceftazidime (30 mg), cefoperazone (75 mg), norfloxacin (10 mg), ciprofloxacin (5 mg), nitrofurantoin (300 mg), furazolidone (300 mg), aztreonam (30 mg), ofloxacin (5 mg) and sulfafurazole (300 mg). The plates were incubated at 28 8C for 18 h and the inhibition of the bacteria by the chemotherapeutic agents was scored. 1.4. Virulence tests of W0702 to tiger frogs Healthy tiger frogs weighing 100 5 g each were held in 100-l tanks supplied with wet pledget at 25–28 8C. Five passages of strain W0702 on frog were performed prior to the virulence tests. The virulence tests with batches of 10 frogs were conducted by intramuscular (IM) injection of bacterial suspensions (1 107 CFU per frog after a 24-h culture) into the leg muscle. Sterile BSS was injected into another group of animals as parallel controls. Each group was duplicated and mortalities were recorded daily for 2 weeks post injection. Reisolation on TSA plates and identification of the bacteria from the brain and the ascites of moribund frogs were performed after bacterial challenge. All animal challenges were carried out following IACUC-approved protocols of Hainan University. 2. Results 2.1. Bacterial isolation Several tens of colonies grew on each plate in all samples after incubation for 24 h on TSA. However, only one type of dominant, white, round colonies and 0.1–0.2 cm in diameter was observed on each plate. Four colonies isolated from each sample were inoculated into TSB for stock culture. All these strains were numbered from W0701 to W0724 (four colonies six frogs). Then the strains W0701, W0702 and W0703 (isolated from the brains of three different frogs) and strains W0704, W0705 and W0706 (isolated from three other frogs) were selected for the following experiment, because the common clinical signs were lethargy, torticolis (presumed brain damage) and cataract (eye damage). 2.2. Bacterial identification The nearly complete 16S rRNA gene sequences of strains W0701 (EU128744), W0702 (EU128742) and
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Z.-Y. Xie et al. / Veterinary Microbiology 138 (2009) 140–144
Table 1 Main phenotypic traits of strains W0701–W0706 and E. meningoseptica. Test methods
Characteristics
Strains W0701–W0706
E. meningoseptica
Microscope
Gram stain Shape
Straight rod
Rods with parallel sides and rounds ends
Growth at
4 8C 25 8C 35 8C 37 8C 40 8C 42 8C
+ + +
NA NA NA + NA
Color
+ Green
NA NA
Color Colony morphology Swarm
+ White 0.1–0.2 cm/round/shiny/raised
+ NA NA
Color Colony morphology
da White 0.1–0.2 cm/round/shiny/raised
db White to yellowb 0.1–0.2 cm/round/shiny
+
db b
Growth on TCBS
Growth on TSA
Growth on McConkey agar
Citrate utilization Malonate utilization
Reactions
Reduction of
NO3 NO2
d
Hydrolysis of
Starch ONPGb Esculin
+ +
+ +
Production of
Indole H2S Gelatinase Urease Oxidase Lysine decarboxylase Ornithine decarboxylase Arginine dihydrolase
+ + + +
d b + d + NA NA NA
Acid production from
Cellobiose Lactose Glucose Sucrose Raffinose Arabinose Rhamnose Trehalose Xylose Fructose Maltose Galactose Mannitol Adonitol Salicin Inositol Aescinate glycoside Sorbitol
+ +
d d d +b +b NA d NA NA NA
ONPG hydrolysis is represent for b-galactosidase production. NA, No data available. a Variable reactions are positive for W0701, W0703 and W0704. b Data from Kim et al. (2005).
W0703 (EU128743) were obtained. They showed 98.6– 98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL reported in Genbank.
All investigated biochemical reactions are shown in Table 1. Briefly, W0701–W0706 were Gram-negative, straight rods, non-motile, positive for oxidase, gelatinase, urease and indole, negative for lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase and H2S.
Z.-Y. Xie et al. / Veterinary Microbiology 138 (2009) 140–144 Table 2 Sensitivity of strain W0702 to various chemotherapeutic agents. Chemotherapeutic agents
Disc content (mg)
Sensitivitya
Ampicillin Streptomycin Erythromycin Chloramphenicol Tetracycline Clindamycin Kanamycin Gentamicin Vancomycin Azitromycin Cefuroxime Ceftazidime Cefoperazone Norfloxacin Ciprofloxacin Nitrofurantoin Furazolidone Aztreonam Ofloxacin Sulfafurazole
10 10 15 30 30 2 30 10 30 15 30 30 75 10 5 300 300 30 5 300
R R R R R R R R S R R R MS R R R R R R R
a
R: Resistant; S: sensitive; MS: moderately sensitive.
They could hydrolyse ONPG and esculin but not starch. Nitrate and nitrite were not reduced as electron acceptors and citrate was not used as a carbon source. Among the 14 saccharides tested, acid was produced aerobically only from glucose and aescinate glycoside. Green colonies were produced on thiosulfate citrate bile salt sucrose agar (TCBS, Difco, USA). Growth on McConkey agar (Difco, USA) was strain depended: only strains W0701, W0703 and W0704 could grow on this medium. No growth occurred at 4 or 40 8C after 2 weeks. All biochemical characteristics tested on strains W0701–W0706 investigated above were consistent with those of E. meningoseptica strains (Holt et al., 1999; Kim et al., 2005) except that acid was not produced from fructose and maltose and that malonate was not used. On the basis of these results, strains W0701–W0706 which are representatives of all the 24 isolates were identified as E. meningoseptica strains. 2.3. Sensitivity of strain W0702 to various chemotherapeutic agents The results of the sensitivity tests of strain W0702 to 20 different chemotherapeutic agents are shown in Table 2. The organism was only susceptible to vancomycin and moderately susceptible to cefoperazone. 2.4. Virulence of strain W0702 to tiger frogs Bacterial cells of strain W0702 were injected into tiger frog and the results showed that W0702 was lethal to tiger frogs. The mortality rate of the infected tiger frog was 80%. Three days following injection with strain W0702, most tiger frogs exhibited the same clinical signs (opaque lens, ascites, torticollis and lethargy) as those observed in naturally infected tiger frogs. Strain W0702 could be reisolated as pure colonies from the brains of all injected frogs and the re-isolated strain was also strongly virulent to healthy tiger frogs.
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3. Discussion All the six isolates (strains W0701–W0706, representatives of the 24 similar strains) obtained from the diseased frogs with typical clinical signs of cataract disease were identified as E. meningoseptica according to their 16S rRNA gene sequences and biochemical characteristics. The few phenotypic discrepancies (shown in Table 1) between the isolates and E. meningoseptica strains were strain dependent. The 16S rRNA gene sequences of strains W0701, W0702, W0703 showed the highest similarity with that of E. meningoseptica strain NTU 870424-IL (accession number is AY468477) whose 16S rRNA gene showed 98.5% similarity with that of E. meningoseptica ATCC 13253. Interestingly, this strain had been isolated from the internal organs of a cultured bullfrog (Rana catesbeiana) with haemorrhagic septicaemia in Taiwan in 1986 (Bernardet et al., 2005). E. meningoseptica was previously reported as the aetiological agent of disease in South African clawed frogs (Xenopus laevis) and leopard frogs (Rana pipiens) (Taylor et al., 1993; Green et al., 1999). The virulence test showed that W0702 was strongly virulent to tiger frog. This is the first report of E. meningoseptica as a pathogen for tiger frog. Certainly, the concurrent infection with this bacterium and virus was possible because no virus test was performed in the study. The strains W0701, W0702 and W0703 were isolated from the brains of diseased frogs, suggesting that E. meningoseptica could break through blood–brain barrier and damage the nervous system. Therefore, the common symptom of diseased frog was torticollis. Neurological signs were considered secondary lesions in bullfrog infected by E. meningoseptica (Mauel et al., 2002). However, they seemed to be primary in tiger frogs as the first clinical sign was torticollis. Although no human case has been reported so far among the staff of frog farms in Hainan, E. meningoseptica is mostly pathogenic for premature or newborn infants and for immunocompromised adults (Bernardet et al., 2006). Since we have frequently isolated E. meningoseptica from the water of frog ponds in Hainan in recent years, it seems that appropriate aquacultural management is essential to avoid this constant threat to frog farming and/or to the staff of frog farms. Antibiograms showed that strain W0702 was resistant to all antibiotics tested except to vancomycin and cefoperazone, which meant that this frog’s pathogen had obtained many antibiotics genes in the long-term evolution process. This explains why this epidemic disease of tiger frog in Hainan was very difficult to control by antibiotic treatment. Hence, a specific vaccine is desirable to control the frog’s disease caused by E. meningoseptica. Acknowledgements This work was supported by Program for New Century Excellent Talents in University, no. NCET-05-0755; grants from the National Natural Science Foundations of China, no. 30660144 and no. 30760190; the Key Project for International Cooperation, no. 2005DFA30610 and the Natural Science Foundations of Hainan Province, no. 80617.
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