DOI: https://doi.org/10.52973/rcfcv-e32191
Received: 16/08/2022 Accepted: 22/09/2022 Published: 28/09/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32191, 1 - 5
ABSTRACT
Rainbow Trout (RT) Oncorhynchus mykiss Walbaum,1792 the rst sh
species to be grown intensively in Turkey and the most commonly
grown species in dams and lakes today. Aeromonas spp. are found in
ecosystems such as freshwater and coastal waters and cause disease
in sh. In RT causes death that is characterized by internal organ
hemorrhages, such as the spleen, kidney and liver and body surface,
ulcerations and congestion. The present study aimed to detect
Aeromonas media bacteria in sh samples collected from the RT farms
in the South Eastern Anatolia Region of Turkey through bacteriological
culturing and MALDI-TOF. Also, it was purposed to determine the
antibiotic susceptibility of the agent. Antibiotic susceptibility testing
was performed using the Kirby–Baüer disk diffusion method. Separate
visits were made to Trout farming facilities in Diyarbakır, Adıyaman,
Şanlıurfa, and Batman in January and February 2021, and 30 sh
with an average live weight of 200–250 grams (g) were collected
from each facility. A total of 1,200 samples were taken from 40 Trout
farms. It was detected the causative agent in the liver, kidney, and
tissue samples of sh from 28 of the 40 farms. The sensitivity of
Enrooxacin (10microgram -µg-), Florfenicol (30µg), Neomycin
(10µg), Amoxicillin (10 µg), Oxytetracycline (30 µg), Erythromycin
(10 µg), Gentamycin (10 µg) and Streptomycin (10 µg) were dened at
chancing ratios. In conclusion, these bacteria were detected in local
farms. These farms must implement measures to minimize stress
factors affecting the sh and avoid overstocking. The best methods
of protection against infection include avoiding overstocking, low
oxygen levels, and unskilled labor.
Key words: Aeromonas media; MALDI-TOF; Oncorhynchus mykiss
RESUMEN
La trucha arcoíris (TA) Oncorhynchus mykiss Walbaum,1792 es la
primera especie de pez que se cultiva de forma intensiva en Turquía
y la especie que se cultiva más comúnmente en presas y lagos en
la actualidad. Aeromonas spp. se encuentran en ecosistemas tales
como agua dulce y aguas costeras y causan enfermedades en los
peces. En la TA provoca la muerte que se caracteriza por hemorragias
en órganos internos, como el bazo, riñón e hígado y superficie
corporal, ulceraciones y congestión. El presente estudio tuvo como
objetivo detectar la bacteria Aeromonas media en muestras de peces
recolectados de las granjas de TA en la región de Anatolia del sureste
de Turquía, a través del cultivo bacteriológico y empleo de MALDI-TOF.
Asimismo, se propuso determinar la susceptibilidad antibiótica
del agente. Las pruebas de susceptibilidad a los antibióticos se
realizaron utilizando el método de difusión en disco de Kirby-Baüer.
Se realizaron visitas separadas a las instalaciones de cultivo de T
en Diyarbakır, Adıyaman, Şanlıurfa y Batman, en enero y febrero
de 2021, y se recolectaron 30 peces con un peso vivo promedio de
200 a 250 gramos (g) en cada instalación. Se tomaron un total de
1.200 muestras de 40 criaderos de T. Se detectó el agente causal en
muestras de hígado, riñón y tejido de peces en 28 de las 40 granjas.
La sensibilidad de enrooxacina (10 microgramo -µg-), orfenicol
(30 µg), neomicina (10 µg), amoxicilina (10 µg), oxitetraciclina (30µg),
eritromicina (10 µg), gentamicina (10 µg) y estreptomicina (10 µg) se
denieron en proporciones de probabilidad. En conclusión, estas
bacterias fueron detectadas en granjas locales. Estas granjas deben
implementar medidas para : minimizar los factores de estrés que
afectan a los peces y evitar el exceso de población. Los mejores
métodos para protegerse contra la infección incluyen : evitar el exceso
de existencias, los bajos niveles de oxígeno y la mano de obra poco
calicada.
Palabras clave: Aeromonas media; MALDI-TOF; Oncorhynchus mykiss
Investigation of diseases caused by Aeromonas media in rainbow trout
(Oncorhynchus mykiss) in commercial sh farms using MALDI-TOF and
specication of antibiotic sensitivity proles of the agent
Investigación de enfermedades causadas por Aeromonas media en truchas arcoíris
(Oncorhynchus mykiss) en piscifactorías comerciales utilizando MALDI-TOF y especicación de
perles de sensibilidad antibiótica del agente
Filiz Özcan*
Dicle University, Veterinary Faculty, Fisheries and Fisheries Diseases Department, Diyarbakır, Turkey.
Email: felizozcan@gmail.com
FIGURE 1. (A) Colony morphology and (B) Spectrum images of
MALDİ-TOF of Aeromonas media
A
B
MALDI-TOF in investigation of diseases caused by Aeromonas media in rainbow trout / Özcan __________________________________________
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INTRODUCTION
Health authorities worldwide emphasize the health benets of
sh and recommend sh consumption at least two times per week
(wk) as part of a healthy diet. While the World Health Organization
(WHO) states that a daily intake of 250–300 miligrams (mg) of
Eicosapentaenoic acid + Docosapentaenoic acid is benecial, the
American Heart Association states that it is necessary to consume
340 grames (g) of sh per wk [27].
Global aquaculture production in 2020 reached a record 122.6 Mil
Ton, including 87.5 Mil Ton of aquatic animals worth USD 264.8 Bill
and 35.1 Mil Ton of algae worth USD 16.5 billion. Around 54.4 Mil Ton
were farmed in inland waters and 68.1 Mil Ton came from marine
and coastal aquaculture [12]. Recently, with the great efforts of
aquaculture farmers, Turkey has become one of the major producers
of aquaculture both in Europe and globally. According to TURKSTAT
data, aquaculture production increased by 12.9% in 2020 compared
to 2019, with Trout (T) being the most important sh species [26].
With the increase in aquaculture production, bacterial diseases
are among the leading causes of losses due to sh diseases under
intensive aquaculture conditions. The most common infective
causative agents include bacteria, such as Aeromonas spp.
Renibacterium salmoninarum, Flavobacterium spp., Yersinia ruckeri,
and Vibrio spp. Among these bacterial sh pathogens, the genus
Aeromonas is notable for having a large number of species [6].
Commercial Rainbow Trout (RT) farming throughout Turkey is
carried out in different water temperatures, stream types (e.g., dams,
spring water, or rivers), and breeding conditions. Aeromonas spp.
an opportunistic pathogen, causes infections in cases of sudden
changes in water temperature and increased stress factors. Although
motile Aeromonas spp., the causative agent of motile Aeromonas
septicemia is not pathogenic to sh, it is responsible for various
infectious complications that have resulted in mortality and outbreaks
in humans [10]. Clinical signs of shes infected by these pathogens
include hemorrhage and ulcer formation on the skin, darkening of the
skin, internal hemorrhages, enlargement of the spleen, exophthalmos
of one or both eyes, n rot, uid accumulation, swimming disorders,
and loss of appetite, with high mortality rates reported [28].
In this study, samples were collected from different T farming
facilities in Diyarbakır, Şanlıurfa, Adıyaman, and Batman Provinces
to investigate A. media. These samples were transported to
the laboratory through cold chain. Following the macroscopic
examination, isolation and identication were performed using culture
and MALDI-TOF detection, respectively and antibiotic susceptibility
was determined. To ease fast and reliable identication of aquatic and
piscine bacteria, matrix assisted laser desorption/ionization time-of-
ight mass spectrometry (MALDI-TOF) [14] has lately been presented
as a useful method instrument [25]. It allows high throughput,
sensitive and specic applications in clinical diagnostics and proling
of bacteria to the genus, species and even strain-level [24]. The main
purpose of the study was to reduce losses by diagnosing and treating
sh diseases correctly.
MATERIALS AND METHODS
Samples were collected from 40 different commercial RT facilities
in Diyarbakır, Adıyaman, Şanlıurfa, and Batman Provinces in the South
Eastern Anatolia Region. Thirty sh with an average live weight of
200–250 g; some clinical signs such as n rot, lesions and ulcers on
the skin, skin darkening, and exophthalmos were considered and
unresponsive sh to feeding and immobile in a certain part of the
pool or oating on the water surface were sampled from each farm.
In total, 1,200 samples were taken to the laboratory for macroscopic
examination before being dissected for internal organs evuluation.
Liver, kidney, and tissue samples obtained from dissection were
inoculated into the medium to identify bacteria. Following the
macroscopic findings, necropsies of the fish delivered to the
laboratory in the cold chain were performed and samples were taken
with sterile swabs. Swab samples were rst transferred to MacConkey
Agar for self-enrichment. The MacConkey Agar (MAC) medium was
used for bacteria isolation seeded plates were incubated for 24 hours
(h) at 28°C, and the dominant uniform bacterial colonies were puried
by streaking three times onto MAC plates [2]. Then, the causative
agents were identied using MALDI-TOF mass spectrometry by Maldi
Biotyper (Bruker, Daltonics, Germany) based on Gram staining and
culture growth proles (FIG. 1).
FIGURE 2. (A) White arrow: Red and petechial hemorrhages on the skin. (B) and (C) White arrows: Erythema and hemorrhages
on the gill and n
CA
B
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Antibiotic susceptibility testing was performed using the Kirby–
Baüer disk diffusion method, Müeller–Hinton medium, and the method
described by Bauer et al. [4]. and evaluated according to the procedures
reported by the Clinical and Laboratory Standards Institute (CLSI) [8],
Ruangpan and Tendencia [22] and Becton, Dickinson and Company [7].
RESULTS AND DISCUSSION
In the present study, A. media were culture isolated and MALDI-TOF
identied from sh with suspected disease from 40 different RT
(Oncorhynchus mykiss) In 28 out of 40 facilities, 1,200 (30 sh/40
farm) samples with stagnation, anorexia, exophthalmos, swimming
disorders, loss of appetite, darkening and color changes of the skin, and
hemorrhages on the base of ns were determined, and the causative
agent were determined. The causative agent was identied in the
liver, kidneys, and tissues of 840 samples. The clinical signs in infected
sh from which tissue samples were taken for bacterial isolation and
identication included kidney hemorrhage, congestion and pallor in
the liver, erythema and hemorrhage on the gill lamellae, yellow-colored
uid accumulation in the intestines, and tail and n rot (FIG. 2).
It was determined that 0-100% of the T farms in the South Eastern
Anatolia Region were contaminated with A. media, and the agent
develops varying levels of resistance to antimicrobial agents. The
incubation period of the disease varies depending on environmental
conditions, care and feeding conditions, age, species and immunity,
accompanying infections, virulence, number, and route of entry of
microorganisms. A larger number of bacteria were isolated from T
farms with low water ow, overpopulation, and turbid pool water.
Antibiogram test results revealed sensitivity to Enrofloxacine,
Florfenicol, and Gentamycine (TABLE 1).
Global population growth and nutritional issues emphasize the
value of aquaculture products (AP) as a high-protein source. To meet
a signicant portion of the protein demand, it is crucial to maintain
the sustainability of aquatic foods and increase the production of AP
in inland and outer waters. The continued expansion of aquaculture
production is regarded as a key strategy for ensuring global food and
nutritional safety and closing the “sh gap,” which is the disparity
between seafood supply and demand [13].
TABLE 1
Antimicrobial susceptibility of Aeromonas media
Antimicrobial agent Isolate 1 Isolate 2 Isolate 3
Enrooxacin (10 µg)
S S S
Florfenicol (30 µg)
S S S
Neomycin (10 µg)
R R R
Amoxicillin (10 µg)
R R R
Oxytetracycline (30 µg)
R R R
Erythromycin (10 µg)
R R R
Gentamycin (10 µg)
S S S
Streptomycin (10 µg)
R R R
µg: microgram. S: Susceptible, R: Resistant
MALDI-TOF in investigation of diseases caused by Aeromonas media in rainbow trout / Özcan __________________________________________
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Diseases of farm-raised sh have become more common with
the development of aquaculture, and facilities have begun to take
hygienic and protective measures to control diseases. Excessive
use of chemicals and antibiotics may result in higher levels of their
residues in sh meat and nature and could also lead to sh immune
system suppression weakening immunity against pathogens [9].
Accordingly, proper bacterial detection and antibiotic use are
required. The genus Aeromonas is responsible for acute, subacute,
chronic, or latent infections in sh, birds, mollusks, and humans
[3]. It results in hemorrhagic septicemia in brown T (Salmo trutta)
goldsh (Carassius auratus), eel (Anguilla anguilla), carp (Cyprinus
carpio), sweet sh (Plecoglossus altivelis ), RT (Oncorhynchus mykiss)
chinook salmon (Oncorhynchus tshawytscha), tilapia, ( Oreochromis
niloticus) and other farm-raised sh species [5].
Allen et al. [1] rst reported A. media isolated from sh pools and
water sources. A. media is a common pathogen found in lakes and
rivers and acts as an intracellular pathogen in sh [13, 17]. In recent
years, A. media has been widely reported. The main ndings include
n rot, hyperemia, abdominal distension and redness [15], and ulcer
formation on the skin [16]. These infectious causative agents of sh
are most prevalent in polluted waters [5]. Bacterial agents in sh
greatly affect sh health and the aquaculture economy, leading to
nancial losses if treatment is not initiated early. Acute infections
cause a high mortality rate in sh, whereas chronic infections have a
low mortality rate [23]. It was determined that 0-100% of T Farms in
the South Eastern Anatolia Region were contaminated with A. media,
and the causative pathogen exhibits varying levels of resistance to
antimicrobial agents.
In present study, the ndings on infected sh were similar to other
studies. Austin and Austin [3] reported redness and inammation of
the ns, widespread bleeding in the anus and skin, swelling of the
eyes and abdomen (ascites), intestinal hemorrhages, and bleeding
and swelling of the tissues such as the spleen and kidneys. In present
study, similar ndings such as bleeding in the kidney, congestion
and pallor in the liver, erythema and hemorrhage on the gill lamellae,
accumulation of yellow-colored uid in the intestines, tail and n rot,
as well as n redness were found (FIG. 2).
The high stock density of sh of different sizes, poor water quality,
and sudden changes in water temperature can cause A. media infections
in sh. Infections due to these bacteria, negatively affect the sh
farming economy, with high mortality in cases of dense sh capacity
[18]. The reasons mentioned above could explain why pathogenic
agents were detected in 28 out of 40 facilities in the present study. The
cleaning conditions of the pools were extremely poor in the aquaculture
facilities where the pathogenic agent was detected. Poor hygiene and
cleaning may have led to poor water quality and the rapid spread of
infection. A higher number of bacteria were isolated from facilities
where the pool water was turbid and polluted.
Previous studies have also shown that factors such as care,
nutrition, changes in the physical and chemical structure of water,
decreased oxygen content, and water pollution influence the
emergence and spread of the disease [11]. Disease recurrence can be
prevented by improving water conditions, avoiding excessive stocking
density, and taking protection and control measures.Treatments that
are not specic to the pathogenic agent do not cure the diseases,
and nancial losses and adverse consequences occur as a result
of inappropriate drug and chemical use [20]. Another issue is that
antibiotics are harmful to the environment, and inappropriate use may
lead to drug resistance in sh [21]. In the literature, increased bacterial
resistance to drugs in aquaculture farms has been reported [15, 24].
Accordingly, testing antibiotic susceptibility is critical to providing
guidance for treatment. In the present study, antibiogram test results
revealed Enrooxacine (10 µg), Florfenicol (30 µg) and Gentamycine
(10 µg) susceptibility. The results of antibiotic susceptibility in the
study of Lü et al. [19]. on A. media is in line with the present ndings.
CONCLUSION
Pathogen positive was found in 840 of the samples examined.
Among the tested antibiotics, Enrooxacin (10 µg), Florfenicol (30
µg), Gentamycin (10 µg) were found to be sensitive to the pathogen.
Appropriate care and feeding conditions, improved water quality,
reduced stock intensity, and the removal of dead sh can reduce
disease emergence and result in faster treatment results. Attention
should be paid to cleaning and hygiene. With regular pool cleaning,
both fecal and feed wastes should be removed; water circulation
should be ensured, and sh scoops, buckets, and nets should be
cleaned. Furthermore, water temperature and oxygen levels should
be monitored regularly.
Conicts of Interest
The authors declare that they have no conicts of interest in the
research.
BIBLIOGRAPHIC REFERENCES
[1] ALLEN, D.A.; AUSTIN, B.; COLWELL, R.R. Aeromonas media,
a new species isolated from river water. Intern. J. System.
Bacteriol. 33(3): 599–604. 1983.
[2] AUSTIN, B. Methods for the diagnosis of bacterial sh diseases.
Marine Life Sci. Tech. 1: 41–49. 2019.
[3] AUSTIN, B.; AUSTIN, D.A. Aerobic Gram-Positive Rods and Cocci.
Bacterial sh pathogens: disease of farmed and wild sh. 6
th
.
Ed. Springer, Dordrecht. 552 pp. 2016.
[4] BAUER, A.U.; KIRBY, W.M.; SHERRIS, J.C.; TRACK, M. Antibiotic
susceptibility testing by standardized single disc method. J.
Clin. Pathol. 45(4): 493–496.1966.
[5] BALTA, F. Fırtına Deresindeki Gökkuşağı Alabalık Çiftliklerinde
izole edilen Aeromonas spp. İzolatlarının Antimikrobiyel
Hassasiyetin Belirlenmesi. J. Anatolian Environm. Anim. Sci.
5(3): 397–407. 2020.
[6] BALCI, Ş. Aeromonas hydrophila ve Aeromonas sobria
Bakterilerinin tanimlanmasinda farkli fenotipik tanimlama
kitlerinin kullanilabilirliğinin karşilaştırılması, Recep Tayyip
Erdoğan Üniversitesi Fen Bilimleri Enstitüsü Su Ürünleri Anabilim
Dali Yüksek Lisans. Tezi. 31 pp. 2020.
[7] BECTON-DICKINSON AND COMPANY (B.D). Antimicrobial
susceptibility test discs. Loveton Circle Sparks, USA. Pp 16. 2011.
[8] CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI).
Methods for antimicrobial disk susceptibility testing of bacteria
isolated from aquatic animals; proposed guideline. M42-P, CLSI,
Wayne, PA. 43pp. 2004.
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32191, 1 - 14
5 of 5
[9] ONUK, E.E.; ÇAYCI, Y.T.; ÇOBAN, A.Y.; ÇİFTCİ, A; BALTA, F.;
DİDİNEN, B.I.; ALTUN, S. Balık ve yetiştirme suyu kökenli
Aeromonas izolatlarının antimikrobiyal duyarlılıklarının saptanması.
Ankara Üniv. Vet. Fak. Derg. 64: 69–73. 2017.
[10] DUMAN, M. Gökkuşaği alabaliklarinda görülen motil aeromonas (A.
hydrophila, A. sobria, A. caviae), Yersinia ruckeri ve Lactococcus
garvieae bakterilerinin antimikrobiyal duyarliliklari ve duyarlilikta
rol oynayan genlerin tespiti, Uludağ Üniversitesi Sağlik Bilimleri
Enstitüsü Su Ürünleri Hastaliklari Anabilim Dali. Doktora Tezi.
116 pp. 2017.
[11] ERER, H. Balık Hastalıkları. 2th. Ed. S. Ü. Basımevi, Konya. 133
pp. 2002.
[12] FOOD AGRICULTURE ORGANIZATION OF THE UNITED NATIONS
(FAO). The State of World Fisheries and Aquaculture, Food,
Agriculture. 2022. On line: https://bit.ly/3rbD1VU. 18/04/2022.
[13] FECKANINOVA, A.; KOSCOVA, J.; MUDRONOVA, D.; POPELKA, P.;
TOROPILOVA, J. The use of probiotic bacteria against Aeromonas
nfections in salmonid aquaculture. Aquacult. 469: 1–8. 2017.
[14] FREIWALD, A.; SAUER, S. Phylogenetic classification and
identification 462 of bacteria by mass spectrometry. Nat.
Protoc. 4: 732–742. 2009.
[15] HUANG, W.M.; WANG, L.; JI, G.Z.; WEI, X.; WANG, H.J.; ZHAO,
J.; GOU, X.L; LONG, Z.M. Identication and drug sensitivity of
bacteria Aeromonas veronii and A. media isolated from Chinese
sucker Myxocyprinus asiaticus. Fisheries Sci. 32: 210–214. 2013.
[16] IGBINOSA, I.H.; IGUMBOR, E.U.; AGHDASI, F.; TOM, M.; OKOH, A.L.
Emerging Aeromonas Species Infections and Their Signicance
in Public Health. Sci. World J. 2012: 1–13. 2012.
[17] KENZAKA, T.; NAKAHARA, M.; HIGUCHI, S.; MAEDA, K; TANI,
K. Draft genome sequences of Amoeba-resistant Aeromonas
spp. isolated from aquatic environments. Genome Announc.
2(5): e01115–14. 2014.
[18] KILINÇ, B. Su ürünlerinde Aeromonas ve Plesiomonas cinsi
mikroorganizmalar ve etkileri. Ege J. Fisheries Aquatic Sci.
36(2): 191–199. 2019.
[19] LÜ, A.J.; HU, X C.; LI, L.; SUN, J.F.; SONG, Y.J.; PEI, C.; ZHANG,
C.; KONG, X.H. Isolation, identification and antimicrobial
susceptibility of pathogenic Aeromonas media isolated from
diseased Koi carp (Cyprinus carpio koi). Iranian J. Fisheries Sci.
15(2): 760–774. 2016.
[20] ÖZTÜRK, N. Bolu ilindeki alabalık üretim tesislerinde kullanılan
ilaç ve kimyasalların araştırılması, Kocaeli Üniversitesi Fen
Bilimleri Enstitüsü Su Ürünleri Anabilim Dalı Yüksek Lisans.
Tezi. 39 pp. 2022.
[21] HILBORN, R.; QUINN, T.; SCHINDLER, D.; ROGERS, D. Biocomplexity
and sheries sustainability. Proceed. Nat. Acad. Sci. United
States of America. 100(11): 6564–6568. 2003.
[22] RUANGPAN, L.; TENDENCIA, A.E. Laboratory manual of
standardized methods for antimicrobial sensitivity tests for bacteria
isolated from aquatic animals and environment. Aquaculture
Extension Manual No 37. Southeast Asian Fisheries Development
Center, Tigbauan. 5021, Iloilo, Philippines. 54 pp. 2004.
[23] STOSKOPF, M.K. Nutrition and Nutritional Diseases. Fish
Medicine. In: W.B. Saunders. Philadelphia. 882pp. 1993.
[24] SHEN, X.J.; HU, X.C.; LAN, Y.; LI, X.; ZHU, A.H; ZHANG, P.; LV,
A.J. Drug susceptibility testing and isolation and identication
of Pseudomonas uorescens from koi carp (Cyprinus carpio koi).
Fisheries Sci. 33: 443–446. 2014.
[25] TOPIĆ-POPOVIĆ, N.; KAZAZIĆ, S.; BOJANIĆ, K.; STRUNJAK-
PEROVIĆ, I.; ČOŽ-RAKOVAC, R. Sample and culture conditions
effects on MALDI-TOF MS identication of bacteria: A review.
Vet. Glasnik. 2021: 1–8. 2021. https://doi.org/jdrm.
[26] Türkish Statistical Institute (TUİK). 2020. Most important sh
species. On Line: https://bit.ly/3SxF7ef. 22/06/2022.
[27] TÜBA, I.I. Gıda ve Sağlıklı Beslenme Sempozyumu Raporu “Su
Ürünleri ve Sağlık” 2019. Dr. Sahin, K. (Ed.). Ankara. On Line:
https://bit.ly/3LIwJpW. 18/02/2022.
[28] YILDIRIM, F. Afyonkarahisar, Antalya ve Eskişehir’den temin
edilen baliklardan aeromonas salmonicida etkeninin izolasyonu
ve identikasyonu ile antibiyotik duyarliliğinin saptanmasi. Afyon
Kocatepe Üniversitesi Sağlık Bilimleri Enstitüsü Mikrobiyoloji
Ana Bilim Dalı. Degree Thesis. 33 pp. 2022.
