Accepted Manuscript

Kocuria rhizophila and Micrococcus luteus as emerging

opportunist pathogens in brown trout (Salmo trutta Linnaeus,
1758) and rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

Agnieszka Pękala, Ewa Paździor, Jerzy Antychowicz, Alicja

Bernad, Hanna Głowacka, Beata Więcek, Wiktor Niemczuk

PII: S0044-8486(17)31457-6
DOI: https://doi.org/10.1016/j.aquaculture.2017.12.028
Reference: AQUA 632982
To appear in: aquaculture
Received date: 27 July 2017
Revised date: 13 December 2017
Accepted date: 18 December 2017

Please cite this article as: Agnieszka Pękala, Ewa Paździor, Jerzy Antychowicz, Alicja
Bernad, Hanna Głowacka, Beata Więcek, Wiktor Niemczuk , Kocuria rhizophila and
Micrococcus luteus as emerging opportunist pathogens in brown trout (Salmo trutta
Linnaeus, 1758) and rainbow trout (Oncorhynchus mykiss Walbaum, 1792). The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Aqua(2017), https://doi.org/10.1016/j.aquaculture.2017.12.028

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 ACCEPTED MANUSCRIPT

Kocuria rhizophila and Micrococcus luteus as emerging opportunist pathogens in brown

trout (Salmo trutta Linnaeus, 1758) and rainbow trout (Oncorhynchus mykiss Walbaum,

1792).

Agnieszka Pękalaa*, Ewa Paździora, Jerzy Antychowiczb, Alicja Bernadc, Hanna Głowackad,

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Beata Więceka, Wiktor Niemczuke

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a
Department of Fish Disease, National Veterinary Research Institute, Al. Partyzantów 57, 24-

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100 Puławy, Poland; a.pekala@piwet.pulawy.pl

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Retired professor, ul. C. K. Norwida 3/6, 24-100 Puławy, Poland;
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jerzy.antychowicz@gmail.com
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c
Department of Veterinary Hygiene, Al. Warszawska 109, 10-702 Olsztyn, Poland;

zhwryby@olsztyn.wiw.gov.pl
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d
Department of Veterinary Hygiene, Al. Powstańców Wielkopolskich 10, 85-090 Bydgoszcz,
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Poland; bydgoszcz.zhwro@wp.pl

e
Department of Epizootiology and Clinic of Bird and Exotic Animals, Faculty of Veterinary
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Medicine, University of Environmental and Life Sciences, ul. C. K. Norwida 25, 50-375
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Wrocław, Poland; wiktor.niemczuk@up.wroc.pl

Corresponding author:

Agnieszka Pękala, e-mail: A.Pekala@piwet.pulawy.pl

tel.: +48 81 8893375, fax: +48 81 886 25 95,

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National Veterinary Research Institute, Department of Fish Diseases, Al. Partyzantów 57,

24-100 Puławy, Poland.

Abstract

In 2014 and 2016 in Poland a few disease outbreaks caused by Kocuria rhizophila and

Micrococcus luteus were diagnosed in rainbow trout and brown trout. In each of these events,

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abnormal mortality (approximately 50%) was accompanied by pathological changes in external

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tissues and internal organs. Exophthalmia, swollen abdomen, increased skin melanisation as

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well as skin petechiae and focal lesions were often seen in moribund fish. In many fish

inflammation of the intestine, liver congestion, and hemorrhages in the tail part of the muscles
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were also observed. Samples from moribund fish internal organs were inoculated onto blood

agar and trypticase soya agar, and incubated for up to 3–4 days at a temperature of 27°C. In the
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majority of cases uniform growth of bacterial colonies was observed; and sometimes these

bacteria appeared in predominant numbers. The bacteria identifications were performed using
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standard kits (API 20 Staph and Vitek 2). Sequencing was carried out so as to improve the
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biochemical identification of isolated bacteria and the evolutionary relationships of their taxa.
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It was also conducted in order to find the possible source of the fish infection. Comparison of
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our strainsˊ molecular structures with the data available in GenBank showed that Kocuria

rhizophila or Micrococcus luteus had never been isolated from diseased fish before, and that
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our isolates were very similar to the strains which had been isolated from food processing

environments (in the case of Kocuria rhizophila) and from scallops (Micrococcus luteus). The

challenge tests performed with our strains of Kocuria and Micrococcus on rainbow trout in

laboratory aquaria confirmed the three Koch postulates. Antibacterial disc diffusion studies

showed that Kocuria rhizophila and Micrococcus luteus are sensitive to most of the drugs tested

i.e. tetracyclines, β-lactams, a macrolide, and amphenicol. The results of these studies show

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that control of outbreaks of the diseases in rainbow trout or brown trout seems realistic if caused

by Kocuria rhizophila or Micrococcus luteus.

Keywords: Micrococcaceae, Kocuria rhizophila, Micrococcus luteus, bacterial fish diseases,

salmonids

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1. Introduction

Bacteria of the genera Kocuria and Micrococcus belong to the family Micrococcaceae, order

Actinomycetales (Kocur et al., 2006). They are Gram-positive, strictly aerobic coccoids. These

bacteria are common skin and oropharynx commensals in mammals, and they also appear in

various environments, inhabit the soil and may occur in several other ecological niches like

marine sediment, chicken meat, fresh water or food (Becker et al., 2008; Lee et al., 2009; Kim

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et al., 2004; Purty et al., 2013).

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In 2014–2016 some cases of Kocuria rhizophila and Micrococcus luteus infection in

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rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) and brown trout (Salmo trutta morpha

lacustris, Linnaeus, 1758) were noted in freshwater farms located in different parts of Poland.
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Exophthalmia, swollen abdomen, increased skin melanisation with focal skin lesions, and

hemorrhages were usually observed in moribund fish. In post mortem examination

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inflammation of the intestine, liver congestion, and hemorrhages in the tail muscles were

noticed. In each of the infected farms fish mortality was estimated at approximately 50%.
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Bacteriological examinations of samples collected from internal organs of diseased fish

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revealed the presence of Kocuria and Micrococcus as the only potential fish pathogens present.
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Only a few reports of Kocuria sp. and Micrococcus sp. isolation from farmed salmonids are
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available. None of them contains information on the state of the fish’s health or bacteria species.

The aims of this study are to determine the phenotypic and molecular properties of
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Micrococacceae which we isolated from moribund salmonids. A comparison of our results to

those of other investigations concerning bacteria belonging to this family was also made. This

research also sought to detect how Kocuria rhizophila and Micrococcus luteus were introduced

to fish farms and if they have or potentially could have pathogenic properties for fish.

2. Material and methods

2.1 Fish examination

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Thirty rainbow trout (weight from 216 to 310 g) and brown trout (weight from 0.4 to 2.8 g)

exhibiting clinical signs of disease were collected from each of three non-proximate farms

located in the southern (2 farms) and northern (1 farm) parts of Poland. Live fish kept in water

were transported directly to the laboratory within a maximum 6h timeframe. The water

temperature did not exceed 12°C. Each fish was examined clinically, bacteriologically, and post

mortem. The presence of parasites was also confirmed. For that purpose, fresh preparations

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from the skin, gills and intestine epidermis were studied microscopically using 100x and 200x

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magnifications.

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2.2 Bacteria isolation

Fish were washed under tap water in order to eliminate saprophytic microflora from the skin.
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Tissue samples from moribund fish skin, kidney and liver were taken for bacteriological

examination using aseptic scalpels and tweezers. Samples were then diluted in sterile
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phosphate-buffered saline (PBS) in the ratio of 1:1 (w/v), and homogenized. The samples were

inoculated onto agar (Biomed) supplemented with 5% horse blood (BA) and onto trypticase
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soy agar (TSA) (bioMériux). After inoculations, media were incubated at 27°C for 72–96 h,
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and the growth of bacteria was estimated. From plates where dense growth was observed, the
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dominant types of bacterial colony were reisolated on BA and incubated at 27°C for 24–48 h.
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Pure cultures were kept frozen for further studies at −80°C in tryptic soy broth (TSB,

bioMérieux) supplemented with 15% glycerol.

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2.3 Bacteria phenotypic studies

Gram staining and assessment of the morphological characteristics of the collected isolates

were performed before biochemical characterization. Biochemical identification was carried

out using the API system (API 20 Staph and Vitek 2) (bioMérieux) according to the

manufacturer´s instructions. After incubation at 27°C for the proper time (mainly 24–48 h), the

results were interpreted using the Apiwe system (bioMérieux).

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2.4 16S rRNA gene sequence analysis

Two isolates collected from rainbow trout and one from brown trout were used for 16S rRNA

gene sequencing. Total genomic bacterial DNA was extracted using a DNeazy Blood & Tissue

Kit (Qiagen) following the manufacturer’s instructions. Two sets of universal primers: 27F (5'-

AGAGTTTGATCCTGGCTCAG -3') and 1492R (5'- TACGGCTACCTTGTTACGACTT -3')

were used to amplify the 16S rRNA gene (Weisburg et al., 1991). PCRs were conducted in 50

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µL reaction mixtures under the following conditions: preheating at 94°C for 2 min, 30 cycles

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of 95°C for 30s, 56°C for 30s, and 72°C for 1 min; and a final extension step at 72°C for 10

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min. Amplified products (about 1500 base pairs) were purified with USB ExoSAP-IT PCR

Product Cleanup (Affymetrix) and sequenced using a 3730xl DNA Analyzer (Genomed S.A.).
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The products of 16S rRNA gene sequencing of the collected isolates were analyzed using

MEGA 7.0 software, and the similarity between the sequences of tested isolates and those
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available at GenBank was evaluated. The evolutionary distances were computed using the

maximum composite likelihood method (Tamura et al., 2004). Phylogenetic trees were
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constructed using the neighbor-joining algorithm with 1000 bootstrap replicates, using MEGA
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7.0 software.
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2.5 Antimicrobial susceptibility test

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The antimicrobial sensitivity of the collected isolates was tested according to the Kirby–

Bauer antibiotic testing method. The disc-diffusion method was performed on Mueller–Hinton
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agar (Oxoid) according to the recommendation of the Clinical and Laboratory Standards

Institute (CLSI, 2006). The following chemotherapeutics from different groups of drugs were

used: the quinolones were flumequine (UB) (30), enrofloxacin (ENR) (5), and oxolinic acid

(OA) (2); the tetracyclines comprised oxytetracycline (OT) (30) and doxycycline (DO) (30); in

the β-lactams amoxycillin (AML) (25) paired with the combination of amoxicillin and

clavulanic acid (AMC) (30); the sole macrolide was erythromycin (E) (15); the

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aminoglycosides complement was neomycin (N) (30); the sulfonamides consisted of

sulfonamide alone (S3) (300), and sulfonamide with trimethroprim (SXT) (25); and florfenicol

(FFC) (30) was the single selection from the amphenicols. After inoculation, the solid media

were incubated at 27°C for 24 h, and the diameters of the inhibition zones were measured to

estimate the antimicrobial sensitivity of collected isolates. Therapeutic recommendations were

issued based on the antimicrobial sensitivity of collected isolates.

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2.6 Challenge test

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In the challenge test, clinically healthy rainbow trout (70–100 g) were used. Ten randomly

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selected fish were examined bacteriologically, parasitologically, and post mortem. For two

weeks the fish were acclimatized to aquaria conditions. Three groups of rainbow trout were
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chosen for experimental infection with three bacteria strains: KD13/2014 (I) and KD14/2014

(II) of Kocuria rhizophila and AOL/2016 (III) of Micrococcus luteus.

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Each experimental group was divided into two sub-groups consisting of 30 fish, for injection

of two variants of bacterial dilutions: 109 cells mL-1 and 107 cells mL-1. Thus, six experimental
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groups were maintained: Ia, Ib, IIa, IIb, IIIa, and IIIb; and one control group IV was assigned.
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Each isolate was cultured on trypticase soya broth (TSB) for 24 h at 27°C and then centrifuged
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at 1800 g for 15 min. The bacteria cell pellets were suspended in sterile PBS to a final
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concentration of 109 cells mL-1 (Ia, IIa, and IIIa) or 107 cells mL-1 (Ib, IIb, and IIIb). The

bacterial concentration was determined using a spectrophotometer at the wavelength 540 nm.
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Fish were anaesthetized by administration of tricaine methanesulfonate (MS-222) (Sigma) for

2–5 min. and were infected by intraperitoneal (i.p.) injection with 0.5 mL of each dilution of

the bacterial suspensions. The control fish group (group IV) was injected with 0.5 mL of sterile

PBS. The fish were maintained in separate 300 L glass tanks in a flow-through system with

dechlorinated and aerated water. During the experiments the water temperature was maintained

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at 11±2°C. Clinical signs in fish were recorded each day over 21 days. Moribund fish were used

for bacteriological and post-mortem examinations.

The Second Local Ethics Commission in Lublin approved the procedure concerning

experiments on fish under resolution no. 70/2014.

3 Results

2.3 Fish examinations

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Moribund fish examinations revealed exophthalmia, swollen abdomen, increased skin

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pigmentation with focal lesions, and petechiae (Fig. 1). In post mortem examination

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inflammation of the intestine, liver congestion, and hemorrhages in the tail part of the muscles

were observed.
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Microscopic examination showed only a few Ichthophthirius multifilis on the skin.
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2.4 Bacteria isolation

After 72–96 h incubation on BA and TSA media, yellow bacterial colonies, 2–3 mm in
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diameter appeared. Uniform growth of this colony type was observed when material gained
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from rainbow trout was inoculated, and growth to predominance when the material originated
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from brown trout.

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2.5 Bacterial biochemical identification

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All bacteria isolated from moribund rainbow trout and brown trout were Gram-positive cocci

arranged in tetrades, and identified as Kocuria varians (strains KD13/2014 and KD14/2014)

and Micrococcus luteus (AOL/2016) by the API 20 Staph and Vitek 2 biochemical kits

respectively. The studies performed on API 20 Staph showed the generally poor reactivity of

the collected isolate. Kocuria varians showed positive reactions only in glucose and fructose

fermentation and acetoin production. Negative reactions were observed in fermentation of

mannose, maltose, lactose, trehalose, mannitol, xylitol, and melibiose, in urea hydrolysis, and

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with dehydratase of arginine. Nitrates were not reduced to nitrites. In the Vitek system, these

bacteria were identified as Kocuria rhizophila.

Bacteria isolated from brown trout (AOL/2016) were identified in API 20 Staph as

Micrococcus luteus, and similar results were achieved with the Vitek 2 system. Those bacteria

also appeared to be poorly reactive biochemically, showing a positive reaction only with

dehydratase of arginine, in urea hydrolysis, and in fermentation of mannitol and sucrose.

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2.6 Sequencing

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Data from 16S rRNA gene sequencing is presented in Table 1 as well as in Fig. 2. Isolates

KD13/2014 (GenBank accession no. KY522908) and KD14/2014 (KY522911) were identified
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as Kocuria rhizophila (≥98.8% and ≥98.5%, respectively), and similarity between them was

99.4%. These sequences were the most similar to Kocuria rhizophila G5 (LN589845) (99.5%)
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(Table 1). The highest sequence similarity of AOL/2016 (GenBank accession no. KY522979)

(≥99.8%) was to Micrococcus luteus. Similarity of 100% between sequences was observed to
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Micrococcus luteus sequences PW 114 (GenBank accession no. KP969047), S6-19

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(KY496281), and SV21 (GU143803) (Table 1).

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A phylogenetic tree was constructed based on the neighbor-joining method (Fig. 2).
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2.7 Challenge tests

The challenge test on rainbow trout showed up to 75% fish mortality starting from the sixth
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day post infection in the Ia and IIa experimental groups. Exophthalmia, exudate fluid in the

body cavity, hemorrhages in the swim bladder, and kidney edema were observed. In groups Ib

and IIb exophthalmia and kidney edema were noticed. Homogenates from internal organs of

infected fish showed the growth of yellow colonies in monoculture, which were identified as

Kocuria rhizophila using the Vitek 2 system.

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In groups IIIa and IIIb, most serious health disorders were observed from the seventh day

post infection. Darkening of the skin, exudate fluid in the body cavity, and kidney edema were

evident. Mortalities were observed only in group IIIa and did not exceed 35%.

No pathological signs or mortalities were observed in fish in the control group (IV).

2.8 Antimicrobial

The results of evaluating the antimicrobial susceptibility of collected isolates to common

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chemotherapeutics used in aquaculture are presented in Table 2. All bacteria showed a large

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zone of growth inhibition, which corresponded to potential sensitivity of bacteria to the β-

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lactams, macrolide, amphenicol, and tetracyclines. Average inhibition zones were observed for

quinolones (ENR), aminoglycoside (N) and sulfonamide with trimethoprim (SXT). Meanwhile
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Kocuria and Miroccoccus strains appeared to be resistant to the quinolones (UB and OA), and

the sulfonamides (S3) (Table 2). Based on the above results, it was recommended to treat fish
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with amoxycillin with clavulanic acid, as a drug potentially active against Gram positive

bacteria. The effects of therapy were satisfactory by oral administration of 50 mg/kg per day
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for 7 days.
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4 Discussion
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Our recent study has shown that bacteria belonging to the Micrococcus family might be
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pathogenic to farmed rainbow and brown trout. Identical pathological symptoms were observed

in spontaneous outbreaks of Kocuria rhizophila and Micrococcus luteus infection in both trout
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species, as well as in experimental challenges. Koch’s postulates were also fulfilled during these

experiments. Micrococcus luteus was described previously as a factor causing clinical signs of

disease consistent with rainbow trout fry syndrome (RTFS) in salmonids (Austin and Stobie,

1992). In diseased fish, mainly exophthalmia, skin melanisation, pale gills and spleen, and

swollen abdomen and kidneys were observed. The symptoms differed in individual fish species.

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Thus, it might be supposed that new infections caused by saprophytic bacteria will appear more

often in future causing appreciable commercial loses on fish farms.

For the first time Kocuria rhizophila and Micrococcus luteus were proved to be the causes

of serious diseases in freshwater salmonids. Authors of research often isolate these bacteria,

most often Kocuria rhizophila, from internal organs of clinically healthy Baltic cod (Gadus

morhua) (data not shown here). The literature data offered no information regarding the impact

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of Kocuria sp. on fish health status, and very little information on this subject concerning

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Micrococcus sp. Only in one report was Kocuria described as a common saprophyte of the trout

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intestine (Kim et al., 2007). It was also noted that these bacteria inhibit the growth of other

microflora. By virtue of this inhibitory property, Kocuria has been successfully used to control
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Aeromonas salmonicida infection in rainbow trout (Irianto and Austin, 2002). Therefore, this

bacterium is a potential candidate constituent for future probiotic ingredients. Kocuria has
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already been applied in the control of V. anguillarum infections in eels and V. arthritis in

rainbow trout (Sharifuzzaman and Austin, 2010).

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Morphologically, bacteria isolated from internal organs of diseased fish were similar to
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previously described saprophytic forms. Neither the biochemical properties of Kocuria

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rhizophila nor the ones of Micrococcus luteus show any significant differences from those of
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the strains isolated by other authors (Austin and Stobie, 1992; Becker at al., 2008). The Vitek

2 system and API 20 Staph correctly identified bacteria, even to the species level, as
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Micrococcus luteus. Our observations suggest that the isolation of Kocuria rhizophila requires

a longer incubation period than other bacteria most frequently isolated from fish, and only the

time longer than 48h is sufficient. The same finding was made previously by Savini et al.

(2010).

Sequencing was performed because of the frequent problem of biochemical

misidentification of bacterial isolates collected from fish. Genome sequencing was also carried

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out to study the evolutionary relationships of the taxa and to find the possible source of fish

infection. Available data in GenBank showed no strains of Kocuria rhizophila or Micrococcus

luteus isolated from diseased fish. Polish isolates of Kocuria rhizophila form a separate cluster,

and are very similar to strains isolated from a food processing environment in Denmark (Røder,

2015) (Fig. 2). Micrococcus luteus was classified very close to the strains collected from

scallops in Canada (Schulze et al., 2006). Our study fills in the gap in the molecular database

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on Kocuria rhizophila and Micrococcus luteus strains obtained from moribund fish and

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supplements the knowledge concerning its pathogenic properties for salmonids. The results of

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our investigation are indispensable for precise monitoring and control of Kocuria rhizophila

and Micrococcus luteus infections in farmed fish in the future. Due to diagnostic difficulties
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and the lack of knowledge about the influence of these microorganisms on fish health, some

outbreaks could be misidentified. In human medicine, outbreaks of human disease caused by

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Kocuria species are still underestimated (Savini et al., 2010).

The drug resistance of Kocuria rhizophila and Microccocus luteus and methods of their
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treatment have been poorly investigated up to now. According to the literature available, there
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are no specified interpretive criteria for Kocuria rhizophila or Micrococcus luteus, especially
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for clinical purposes (CLSI, 2008). Szczerba (2003) displayed some data concerning the
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antimicrobial resistance of Kocuria, Micrococcus, Nesterenkonia, Kytococcus and

Dermacoccus, but without species specifications. The author showed that these bacteria were
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resistant to erythromycin, which was contrary to our results. However, his results concerning

bacteria susceptibility to doxycycline, and amoxicillin/clavulanate wholly concur with ours.

The roles of Kocuria and Micrococcus species in fish pathology are still uncertain and should

be investigated further. Although the presented studies showed pathogenic properties of these

microorganisms to trout, further supplementary exploration is necessary to fill in this

knowledge gap.

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Conclusions:

1. According to the results of our investigations Kocuria rhizophila and Microccocus

luteus display some pathogenic properties to rainbow trout and brown trout, for which

the explanation may lie in the change in the aquatic environment and its negative

influence on the fish immune system.

2. Our study shows that these bacterial infections in salmonids could potentially be

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controlled by applying adequate chemotherapeutics.

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3. The Kocuria rhizophila and Mircoccocus luteus isolated from rainbow and brown trout

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farmed in Poland show some genomic similarity to organisms which are being isolated

from processing environments.

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4. Information scarcity in GenBank impedes the elucidation of the source of Kocuria

rhizophila and Mircoccocus luteus infections in freshwater Polish salmonids, however

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the genetic similarity of our isolates to bacteria isolated from a food processing

environment suggests that fish food could be the possible source of these bacterial
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infections.
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Funding:
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This research was supported by the KNOW (Leading National Research Centre) Scientific
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Consortium "Healthy Animal - Safe Food", Ministry of Science and Higher Education
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resolution no. 05-1/KNOW2/2015

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References:

1. Austin B., Stobie M., 1992. Recovery of Micrococcus luteus and presumptive

Planococcus from moribund fish during outbreaks of rainbow trout (Oncorhynchus

mykiss Walbaum) fry syndrome (RTFS) in England. J Fish Dis 15, 302–206.

2. Becker K., Rutsch F., Uekotter A., Kipp F., Konig J., Marquardt T., Peters G., von Eiff

PT
C., 2008. Kocuria rhizophila adds to the emerging spectrum of micrococcal species

involved in human infections. J Clin Microbiol 46, 3537–3539.

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3. Clinical and Laboratory Standards Institute, (CLSI), 2006. VET03-A Methods for

SC
antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals;

Approved guideline. vol. 26, no. 23, Wayne, PA, USA.

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4. Clinical and Laboratory Standards Institute (CLSI), 2008. M100-S25 Performance

standards for antimicrobial susceptibility testing. 25th International Supplement, , vol.

MA

35, no. 3. Wayne, PA, USA.

5. Irianto A., Austin B., 2002. Probiotics in aquaculture. J Fish Dis 25, 633–642.
E D

6. Kim D. H., Brunt J., Austin B., 2007. Microbial diversity of intestinal contents and
PT

mucus in rainbow trout (Oncorhynchus mykiss). J Appl Microbiol 102, 1654–1664.

7. Kim S. B., Nedashkovskaya O. I., Mikhailov V. V., Han S. K., Kim K. O., Rhee M. S.,
CE

Bae K. S., 2004. Kocuria marina sp. nov., a novel actinobacterium isolated from marine
AC

sediment. Int J Syst Evol Micr 54, 1617–1620.

8. Kocur M., Kloos W. E., Schleifer K. H., 2006. The Genus Micrococcus. In: Dworkin

M., Falkow S., Rosenberg E., Schleifer K. H., Stackebrandt E. (Eds.), The Prokaryotes

(3rd ed.), Springer, New York, pp. 961–971. doi.org/10.1007/0-387-30743-5_37

9. Lee J. Y., Kim S. H., Jeong H. S., Oh S. H., Kim H. R., Kim Y. H., Lee J. N., Kook J.

K., Kho W. G., 2009. Two cases of perinonitis caused by Kocuria marina in patients

undergoing continuous ambulatory peritoneal dialysis. J Clin Microbiol 47, 3376–3378.

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10. Purty A., Saranathan R., Prashanth K., Narayanan K., Asir J., Devi Ch. S., Amarnath S.

K., 2013. The expanding spectrum of human infections caused by Kocuria species: a

case report and literature review. Emerg Microbes Infec 2, e7; doi:

10.1038/emi.2013.71.

11. Røder H. L., Raghupathi P. K., Herschend J., Brejnrod A., Knøchel S., Sørensen S. J.,

Burmølle M., 2015. Interspecies interactions result in enhanced biofilm formation by

PT
co-cultures of bacteria isolated from a food processing environment. Food Microbiol

RI
51, 18–24.

SC
12. Savini V., Catavitello Ch., Masciarelli G., Astolfi D., Balbinot A., Bianco A., Febbo F.,

DˊAmario C., DˊAntonio D., 2010. Drug sensitivity and clinical impact of members of
NU
the genus Kocuria. J Med Microbiol 59, 1395–1402.

13. Schulze A. D., Alabi A. O., Tattersall-Sheldrake A. R., Miller K. M., 2006. Bacterial
MA

diversity in a marine hatchery: Balance between pathogenic and potentially probiotic

bacterial strains. Aquaculture 256, 50–73.

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14. Sharifuzzaman S. M., Austin B., 2010. Development of protection in rainbow trout
E

(Oncorhynchus mykiss, Walbaum) to Vibrio anguillarum following use of the probiotic

PT

Kocuria SM1. Fish Shellfish Immun 29, 212–216.

CE

15. Szczerba I., 2003. Susceptibility to antibiotics of bacteria from genera Micrococcus,

Kocuria, Nesterenkonia, Kytococcus and Dermacoccus. Med Dosw Mikrobiol 55, 75–
AC

80.

16. Tamura K., Nei M., Kumar S., 2004. Prospects for inferring very large phylogenies by

using the neighbor-joining method. Proc Natl Acad Sci USA 101(30), 11030–11035.

17. Weisburg W. G., Barns S. M., Pelletier D. A., Lane D. J., 1991. 16S ribosomal DNA

amplification for phylogenetic study. Journal of Bacteriol 173, 697–703.

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Table 1. Similarity values of 16S rRNA gene sequences of Kocuria and Micrococcus luteus.

Kocuria rhizophila
G5 DC2201 LE 78 M1B71 NT 92
(LN58984 (KM46093 (FN90844 (LN81298 (KT38733
5) 9) 6) 4) 5)
KD13/201 99.5 98.9 98.8 98.9 98.8
4
(KY52290
8)
KD14/201 99.8 98.6 98.5 98.6 98.5

PT
4
(KY52291
1)

RI
Micrococcus luteus

SC
PW 114 S6-19 SV21 CKS 1011 NCTC Sc-GM1
(KP96904 (KY49628 (GU14380 (KC96825 2665 (DQ35778
7) 1) 3) 4) (NR 3)
075062)
NU
AOL/2016 100 100 100 99.8 99.8 99.8
(KY52297
9)
MA
E D
PT
CE
AC

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Table 2. Antimicrobial susceptibility of collected isolates

Antimicrobial drug and growth inhibition zone size
(mm)
Isolates ENR UB OA OT DO AMC AML E N FFC S3 SXT
KD13/2014 17 6 6 30 30 35 35 35 25 35 6 26
KD14/2014 20 6 6 25 22 35 35 33 20 30 6 12
AOL/2016 17 14 6 18 28 23 28 27 20 28 6 24

PT
RI
SC
NU
MA
E D
PT
CE
AC

18
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Highlights

 This is the first isolation of Kocuria rhizophila, and the second of Mircrococcus luteus

from diseased fish.

 The isolates were genetically and phenotypically characterized, and were found to be

associated with fish health disorders.

 The results suggested that those bacteria need to be taken into consideration as a

PT
potentially pathogenic for fish.

RI
 This study helps further understand the biological challenges of aquatic environment.

SC
NU
MA
E D
PT
CE
AC

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Figure 1
Figure 2