Pathogenic Gram-negative bacteria isolated from ornamental fish in ...

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Abstract. Ornamental fish culture and trade is a major global activity. Bacteria are the main pathogenic micro- organisms affecting fish, generally in the form of ...
Bull. Eur. Ass. Fish Pathol., 33(6) 2013, 181

Pathogenic Gram-negative bacteria ’œ˜•ŠŽȱ›˜–ȱ˜›—Š–Ž—Š•ȱ꜑ȱ’—ȱ›žžŠ¢DZȱ characterization and antibiotic resistance ǯȱŠ›—ŽŸ’ŠȘǰȱǯȱŽŠ–Ž—ÇŠȱŠ—ȱǯȱŽ››ŽĴŠ Departamento de Acuicultura y Patología de Organismos Acuáticos, Facultad de Veterinaria, Universidad de la República, Uruguay

Abstract ›—Š–Ž—Š•ȱ꜑ȱŒž•ž›ŽȱŠ—ȱ›ŠŽȱ’œȱŠȱ–Š“˜›ȱ•˜‹Š•ȱŠŒ’Ÿ’¢ǯȱŠŒŽ›’ŠȱŠ›Žȱ‘Žȱ–Š’—ȱ™Š‘˜Ž—’Œȱ–’Œ›˜˜›Š—’œ–œȱŠěŽŒ’—ȱ꜑ǰȱŽ—Ž›Š••¢ȱ’—ȱ‘Žȱ˜›–ȱ˜ȱ˜™™˜›ž—’œ’Œȱ™Š‘˜Ž—œǯȱ ›Š–Ȭ—ŽŠ’ŸŽȱ‹ŠŒŽ›’Šȱ ˜ž—ȱ˜••˜ ’—ȱŠȱœž›ŸŽ’••Š—ŒŽȱ˜ȱ˜›—Š–Ž—Š•ȱ꜑ȱŠ›–œȱŠ—ȱ™Žȱœ‘˜™œȱ’—ȱ›žžŠ¢ȱŠ›ŽȱŽœŒ›’‹Žǯȱ ȱ˜Š•ȱ˜ȱŜśȱ꜑ȱ Ž›ŽȱŽ¡Š–’—Žȱǻřŗȱ˜•ęœ‘ȱŠ—ȱřŚȱ›˜™’ŒŠ•ȱ꜑Ǽǯȱ œ˜•ŠŽœȱ Ž›Žȱ™‘Ž—˜¢™’ŒŠ••¢ȱ ’Ž—’ꮍȱžœ’—ȱ‘Žȱ’Œ›˜‹ŠŒȱŘŖŖŖȱ”’ȱǻ ǼȱŠ—ȱŠ—’‹’˜’ŒȱœŽ—œ’’Ÿ’’Žœȱ Ž›ŽȱŠœœŽœœŽȱžœ’—ȱ ¡˜’ȱȱŽ—œ’Ȭ’œŒœǯȱȱ•ŽŠœȱŘŖȱ‹ŠŒŽ›’Š•ȱœ™ŽŒ’Žœȱ Ž›Žȱ’Ž—’ꮍǰȱ ’‘ȱAeromonas, Pseudomonas and Vibrioȱ‘Žȱ–˜œȱ›ŽšžŽ—ȱŽ—Ž›ŠǯȱŽœ˜™‘’•’Œȱ–˜’•ŽȱAeromonas spp.  Ž›Žȱ‘Žȱ–˜œȱ›ŽšžŽ—•¢ȱ ’Ž—’ꮍȱ’œ˜•ŠŽœǯȱ’—’ęŒŠ—ȱ’쎛Ž—ŒŽœȱŒ˜—ŒŽ›—’—ȱŠ—’‹’˜’ŒœȱœŽ—œ’’Ÿ’¢ȱ Ž›Žȱ˜ž—ǰȱŠ—ȱ‘Ž¢ȱ were divided into two groups: those with high sensitivity (chloramphenicol; tetracycline, oxytetracycline, oxolinic acid and sulphamethoxazole/trimethroprim), and those with low sensitivity ǻ™Ž—’Œ’••’—ȱŠ—ȱŠ–™’Œ’•’—Ȧœž•‹ŠŒŠ–Ǽǯȱž•’Ȭ›Žœ’œŠ—ŒŽȱ Šœȱ˜‹œŽ›ŸŽȱ’—ȱŚśƖȱ˜ȱ‘Žȱ’œ˜•ŠŽœǯȱ••ȱ‘ŽœŽȱ ˜›Š—’œ–œȱ•’”Ž•¢ȱ™˜œŽȱŠȱ•’–’Žȱ›’œ”ȱ˜ȱ‘ž–Š—œǰȱ˜›—Š–Ž—Š•ȱ꜑ȱ‘Š—•Ž›œȱœ‘˜ž•ȱ‹Žȱ Š›—Žȱ˜ȱ‘Žȱ potential zoonotic risks involved.

Introduction ›—Š–Ž—Š•ȱ꜑ȱŒž•ž›ŽȱŠ—ȱ›ŠŽȱ’œȱŠȱ–Š“˜›ȱ

˜’Œœȱ˜›ȱŠšžŠŒž•ž›ŽȱŠ—ȱŒ˜–™Š—’˜—ȱŠ—’–Š•œȱ’œȱ

activity with greater than 1 billion animals per ¢ŽŠ›ȱ›ŠŽȱ•˜‹Š••¢ȱǻ‘’Ĵ’—˜—ȱŠ—ȱ‘˜—ǰȱ

restricted in most countries (FDA, 2012; Costello

ŘŖŖŝǼǯȱ›—Š–Ž—Š•ȱ꜑ȱŠ›ŽȱŽ¡™˜œŽȱ˜ȱ–Š—¢ȱ

subjected to treatments without a veterinary

œ›Žœœ˜›œǰȱ–Š’—•¢ȱŠœȱŠȱ›Žœž•ȱ˜ȱ‘Ž’›ȱŒ˜–™•Ž¡ȱ supply chain, making them susceptible to many

prescription. These treatments include the use ˜ȱŠȱ—ž–‹Ž›ȱ˜ȱŠ—’‹’˜’Œœȱ‘ŠȱŒŠ—ȱ‹ŽȱŠ™™•’Žȱ inappropriately. As a result, bacteria isolated ›˜–ȱ˜›—Š–Ž—Š•ȱ꜑ȱ˜Ž—ȱœ‘˜ ȱ–ž•’Ȭ›Žœ’œtance to several antibiotics (Yanong, 2010; del

diseases. Ectoparasites and bacteria are the most œ’—’ęŒŠ—ȱ˜›—Š–Ž—Š•ȱ꜑ȱ™Š‘˜Ž—œǯȱ ›Š–ȱ negative bacteria are the main pathogenic miŒ›˜˜›Š—’œ–œȱŠěŽŒ’—ȱ꜑ǯȱ¡›’—œ’Œȱœ›Žœœ˜›œǰȱ including crowding, shipping and poor water šžŠ•’¢ȱŒŠ—ȱ™›Ž’œ™˜œŽȱ˜›—Š–Ž—Š•ȱ꜑ȱ˜ȱ‹ŠŒŽ›’Š•ȱ’œŽŠœŽœȱǻŽ ‹Š›ǰȱŘŖŖŗǼǯȱ‘ŽȱžœŽȱ˜ȱŠ—’‹’* Corresponding author’s e-mail: [email protected]

ŽȱŠ•ǯǰȱŘŖŖŗǼǯȱ ˜ ŽŸŽ›ȱ˜›—Š–Ž—Š•ȱ꜑ȱŠ›Žȱ˜Ž—ȱ

Rio-Rodriguez and Turnbull, 2002). The deter–’—Š’˜—ȱ˜ȱ‘Žȱ›Žœ’œŠ—ŒŽȱ•ŽŸŽ•œȱ˜ȱ‹ŠŒŽ›’Šȱ˜ȱ ŸŠ›’˜žœȱŠ—’‹’˜’Œœȱ’œȱŠȱ‘Ž•™ž•ȱž’Žȱ˜ȱ‘Ž›Š™¢ǯȱ ȱž›‘Ž›ȱŒ˜—ŒŽ›—ȱ’œȱ‘Šȱœ˜–Žȱ꜑ȱ™Š‘˜Ž—’Œȱ

182, Bull. Eur. Ass. Fish Pathol., 33(6) 2013

‹ŠŒŽ›’Šȱ–Š¢ȱ‹Žȱ£˜˜—˜’Œǯȱ›—Š–Ž—Š•ȱ꜑ȱŠ›Žȱ Šȱ™˜Ž—’Š•ȱ›ŽœŽ›Ÿ˜’›ȱ˜ȱ’—ŽŒ’˜žœȱŠŽ—œȱ˜›ȱ

˜›ȱ ›Š–ȱœŠ’—ǰȱŒ¢˜Œ‘›˜–Žȱ˜¡’ŠœŽȱŠŒ’Ÿ’¢ȱ and motility. Microbact 2000 kit (OXOID) was

 ’•ȱŠ—ȱŠ›–Žȱ꜑ȱŠ—ȱ‘ž–Š—œǯȱ‘’œȱœž¢ȱ ’œȱŠȱŽœŒ›’™’˜—ȱ˜ȱ‘Žȱ ›Š–Ȭ—ŽŠ’ŸŽȱ‹ŠŒŽ›’Šȱ

žœŽȱ˜›ȱ’Ž—’ęŒŠ’˜—ǰȱ ’‘ȱœŠ–™•Žœȱ’—Œž‹ŠŽȱ

˜ž—ȱ˜••˜ ’—ȱœž›ŸŽ’••Š—ŒŽȱ˜ȱ˜›—Š–Ž—Š•ȱ꜑ȱ

‘ŽȱœŽ—œ’’Ÿ’¢ȱ˜ȱŠȱ›Š—Žȱ˜ȱŠ—’‹’˜’Œœȱ¡˜’ȱ

Š›–œȱŠ—ȱ™Žȱœ‘˜™œȱ’—ȱ›žžŠ¢ǯ

BBL Sensi-Discs were used. These included penicillin (P; 10 IU), chloramphenicol (C; 30 μg),

Material and methods

tetracycline (TE; 30 μg), oxytetracycline (OT;

’œ‘ȱ Ž›Žȱ˜‹Š’—Žȱ›˜–ȱ™Žȱœ‘˜™œȱ˜›ȱ˜›—Š–Ž—-

30 μg), Oxolinic Acid (OA; 2 μg), ampicillin/

Š•ȱ꜑ȱŠ›–œǯȱ—•¢ȱ꜑ȱ ’‘ȱŽŸ’Ž—ȱœ’—œȱ˜ȱ

œž•‹ŠŒŠ–ȱǻDzȱŗŖȦŗŖȱΐǼȱŠ—ȱœž•Š–Ž˜¡Š£-

’œŽŠœŽȱ Ž›ŽȱœŠ–™•Žǯȱȱ˜Š•ȱ˜ȱŜśȱ꜑ȱ Ž›ŽȱŽ¡-

ole/trimethroprim (STX; 23.5/1,25 μg). Isolated

Š–’—Žȱǻřŗȱ˜•ęœ‘ȱŠ—ȱřŚȱ›˜™’ŒŠ•ȱ꜑ŽœǼǯȱ‘Žȱ ꜑ȱœ™ŽŒ’ŽœȱŽ¡Š–’—Žȱ Ž›ŽDZȱ˜•ęœ‘ǰȱCarassius auratus; guppy, Poecilia reticulatus; platy, Xipho-

colonies were suspended in saline and adjusted ˜ȱŠȱŽ—œ’¢ȱ˜ȱŖǯśȱŠŒŒ˜›’—ȱ˜ȱŒŠ›•Š—ȱž›bidity standards. TSA plates were inoculated

phorus maculatus; swordtail, Xiphophorus helleri;

by streaking with swab over the entire agar

Molies, Poecilia sphenops and P. latipinna; discus,

œž›ŠŒŽȱŠ—ȱ’—Œž‹ŠŽȱŠȱŘśȱǚȱ˜›ȱŘŚȱ‘ǯȱ˜ž›ȱ discs per plate were used (NCCLS, 2002). CriŽ›’Šȱ˜›ȱŽŽ›–’—’—ȱœžœŒŽ™’‹’•’¢ȱŠ›Žȱœ‘˜ —ȱ ’—ȱŠ‹•ŽȱŗǯȱŠŒŽ›’ŠȱŠ••’—ȱ’—˜ȱ‘ŽȱŒŠŽ˜›’Žœȱ˜ȱ

Symphisodon aequifasciataDzȱ Š›ȱ˜ž›Š–’ǰȱTrichogaster laliusDzȱŠ—ȱ™Ž™™Ž›ŽȱŒŠęœ‘ǰȱCorydoras paleatus. Microbiological samples were taken ›˜–ȱ‘Žȱœ”’—ȱǻ›˜–ȱ꜑ȱ ’‘ȱœ”’—ȱž•ŒŽ›œǼȱŠ—ȱ kidney (in all cases). The samples were plated onto trypticase soy agar (TSA) and incubated ŠȱŘśȱǚȱ˜›ȱŘŚȱ‘ǯȱŽœž•Š—ȱŒ˜•˜—’Žœȱ Ž›ŽȱŽœŽȱ

ŠȱŘśȱǚȱ˜›ȱŚŞȱ‘ȱ™›’˜›ȱ˜ȱŠ—Š•¢œ’œǯȱ˜ȱŽŽ›–’—Žȱ

susceptible or intermediately susceptible were both considered as being susceptible, according to del Rio-Rodriguez and Turnbull (2002). Žœž•œȱ Ž›ŽȱœŠ’œ’ŒŠ••¢ȱŽŸŠ•žŠŽȱžœ’—ȱΛ2ȱ˜›ȱ

Table 1.ȱ›’Ž›’Šȱ˜›ȱŠ—’–’Œ›˜‹’Š•ȱœžœŒŽ™’‹’•’¢ȱŽŽ›–’—Š’˜—ȱ

Antimicrobial agent

Inhibition zone diameter (mm) Interpretative criteria Sensitivity

Source

Resistant

Penicillin (P)

> 20

< 19 NCCLS, 2002

Chloramphenicol (C)

> 13

< 12

Tetracycline (TE)

> 15

< 14 CLSI, 2013

Oxytetracycline (OT)

> 14

< 13

del Rio-Rodriguez & Turnbull, 2002

Oxolinic Acid (OA)

> 17

< 16

del Rio-Rodriguez & Turnbull, 2002

Ampicillin/Sulbactam (SAM)

>12

< 11 CLSI, 2013

ž•Š–Ž‘˜¡Š£˜•ŽȦ›’–Ž‘›˜™›’–ȱǻǼ

> 11

< 10 CLSI, 2013

del Rio-Rodriguez & Turnbull, 2002

Bull. Eur. Ass. Fish Pathol., 33(6) 2013, 183

‘Žȱ™›˜™˜›’˜—œȱ˜ȱ’œ˜•ŠŽœȱœžœŒŽ™’‹•Žȱ˜ȱ‘Žȱ ’쎛Ž—ȱŠ—’‹’˜’Œœǯ

quently isolated species are A. hydrophila, A. caviae and A. veronii (Janda et al, 1995; Lewbart, 2001). Our results showed A. hydrophila -like

Results

isolates were the most dominant TSA-culturable

ȱ˜Š•ȱ˜ȱŜŝȱ’œ˜•ŠŽœȱ Ž›Žȱ˜‹Š’—ŽDZȱřśȱ›˜–ȱ™Žȱ

bacteria recovered (37.3 %). This is similar to ™›ŽŸ’˜žœȱœž’Žœȱ›˜–ȱ›žžŠ¢ȱǻ‘ŠŸŽœȱŽȱŠ•ǯǰȱ

œ‘˜™ȱ꜑ŽœȱŠ—ȱřŘȱ›˜–ȱŠ›–ȱ꜑Žœǯȱȱ˜Š•ȱ˜ȱŘŖȱ ‹ŠŒŽ›’Š•ȱœ™ŽŒ’Žœȱ Ž›Žȱ’Ž—’ꮍȱǻŠ‹•ŽȱŘǼǯȱ‘Žȱ most common bacterial genera were: Aeromonas,

2003; Carnevia et al., 2010) and other countries (Kuo and Chung, 1994; Shome and Shome,

Pseudomonas and Vibrio, with representatives

ŗşşşDzȱŠ—ę—ȱŽȱŠ•ǰȱŘŖŖŘDzȱ˜ŒŠŽ••’ȱŽȱŠ•ǰȱŘŖŖřǼǯȱA.

›˜–ȱŠ••ȱ‘›ŽŽȱŽ—Ž›Šȱ˜ž—ȱ›˜–ȱ‹˜‘ȱ™Žȱœ‘˜™œȱ

hydrophila can be a primary pathogen, however

Š—ȱ꜑ȱŠ›–œȱ’Ž—’ꮍǯȱ‘Ž›Žȱ Šœȱ—˜ȱœ’—’ę-

it is more commonly implicated as a second-

ŒŠ—ȱ’쎛Ž—ŒŽȱ‹Ž ŽŽ—ȱ‘Žȱ—ž–‹Ž›ȱ˜ȱ‹ŠŒŽ›’Š•ȱ Ž—Ž›Šȱ’Ž—’ęŒŠ’˜—œȱ’—ȱŽŠŒ‘ȱ™•ŠŒŽȱǻŠ›–ȱ˜›ȱ™Žȱ

Š›¢ȱ˜™™˜›ž—’œ’Œȱ™Š‘˜Ž—ǰȱ˜Ž—ȱŠœœ˜Œ’ŠŽȱ with poor water quality or a serious errors in

œ‘˜™Ǽǯȱ‘Žȱ–˜œȱ›ŽšžŽ—•¢ȱ’Ž—’ꮍȱ‹ŠŒŽ›’Šȱ was Aeromonas spp. (49.2 % isolates).

production management (Padrós and Furones; 2004). œŽž˜–˜—ŠœȱœžĵŽ›’, P. aeruginosa and P. ̞˜›ŽœŒŽ—œ have been associated with diseas-

—’‹’˜’ŒȱœŽ—œ’’Ÿ’’Žœȱ˜›ȱ‘Žȱ’쎛Ž—ȱ’œ˜•ŠŽœȱ Š›Žȱœ‘˜ —ȱ’—ȱŠ‹•ŽȱŘǯȱ‘Ž›Žȱ Ž›Žȱœ’—’ęŒŠ—ȱ’-

Žœȱ’—ȱŠ›–Žȱ꜑ȱŠ—ȱŒ›žœŠŒŽŠ—œȱǻ˜••ŽȱŠ—ȱ Grischkowsky, 1981; Nabi et al., 2000) as well

Ž›Ž—ŒŽœȱ‹Ž ŽŽ—ȱ‘ŽȱœŽ—œ’’Ÿ’¢ȱ˜ȱ‘Žȱ’œ˜•ŠŽœȱ˜ȱ

Šœȱ˜›—Š–Ž—Š•ȱ꜑ȱǻŠ›”Ž›ǰȱŘŖŖŗDzȱŽ›ŽŽȱŽȱŠ•ǯǰȱ

2

‘Žȱ’쎛Ž—ȱŠ—’‹’˜’ŒœȱǻΛ = 32.39; p