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Antimicrobial Resistance in
Lactococcus
spp. Isolated from Native
Brazilian Fish Species: A Growing
Challenge for Aquaculture
Angélica Emanuely Costa do Rosário , Angelo Carlo Chaparro Barbanti , Helena Caldeira Matos ,
Cynthia Rafaela Monteiro da Silva Maia , Júlia Miranda Trindade , Luiz Fagner Ferreira Nogueira ,
Fabiana Pilarski , Silvia Umeda Gallani , Carlos Augusto Gomes Leal , Henrique César Pereira Figueiredo ,
Guilherme Campos Tavares *
Posted Date: 30 October 2024
doi: 10.20944/preprints202410.2381.v1
Keywords: disk diffusion; epidemiological cut-off values; fish; piscine lactococcosis
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Article
Antimicrobial Resistance in Lactococcus spp. Isolated
from Native Brazilian Fish Species: A Growing
Challenge for Aquaculture
Angélica Emanuely Costa do Rosário 1, Angelo Carlo Chaparro Barbanti 1,
Helena Caldeira Matos 2, Cynthia Rafaela Monteiro da Silva Maia 1, Júlia Miranda Trindade 2,
Luiz Fagner Ferreira Nogueira 2, Fabiana Pilarski 3, Silvia Umeda Gallani 1,
Carlos Augusto Gomes Leal 2, Henrique César Pereira Figueiredo 2 and
Guilherme Campos Tavares 2,*
1 Post-graduate Program in Aquaculture, Nilton Lins University, Manaus, Amazonas, Brazil
2 Department of Preventive Veterinary Medicine, School of Veterinary Medicine, Federal University of
Minas Gerais – UFMG, Belo Horizonte, Minas Gerais, Brazil
3 Laboratory of Microbiology and Parasitology of Aquatic Organisms, São Paulo State University (Unesp),
Aquaculture Center of Unesp, Jaboticabal, São Paulo, Brazil
* Correspondence: gcamposvet@hotmail.com; Tel.: +55-31-34092126
Abstract: Lactococcus spp. has emerged as a pathogen that is affecting global aquaculture, with L. garvieae, L.
petauri, and L. formosensis causing piscine lactococcosis. While antimicrobials are commonly used to treat
diseases in aquaculture, reports of antimicrobial resistance in fish isolates are increasing. However, little is
known about the susceptibility patterns of Lactococcus spp. strains isolated from native fish species in Brazil.
This study aimed to assess the antimicrobial susceptibility of these strains and establish a provisional
epidemiological cut-off value for L. garvieae using the normalized resistance interpretation approach. A total of
47 isolates were tested: 17 L. garvieae, 24 L. petauri, and 6 L. formosensis. The isolates were classified as wild-type
or non-wild-type (NWT) based on inhibition zone diameters. Isolates classified as NWT for three or more
antimicrobial classes were considered multidrug-resistant, and the multiple antibiotic resistance (MAR) index
was calculated. The results revealed heterogeneity in antimicrobial resistance profiles, with higher resistance
to trimethoprim/sulfamethoxazole and norfloxacin. Resistance to other antimicrobials, including florfenicol
and oxytetracycline (approved for use in Brazil), varied according to the bacterial species. Lactococcus petauri
(87.5%) and L. formosensis (66.7%) showed the highest multidrug resistance, compared to L. garvieae (11.7%),
along with higher MAR index values. These findings suggest that multidrug-resistant strains could pose future
challenges in the production of native species, underscoring the need for ongoing monitoring of antimicrobial
resistance and responsible use of antimicrobials in aquaculture.
Keywords: disk diffusion; epidemiological cut-off values; fish; piscine lactococcosis
1. Introduction
Piscine lactococcosis is considered an emerging bacterial disease for fish farming worldwide [1],
and the number of hosts in which Lactococcus garvieae, L. petauri and L. formosensis have been detected
has expanded [2–7]. The disease is currently a significant health challenge for Oncorhynchus mykiss
and Oreochromis niloticus production, causing high mortality rates and significant economic losses [8–
10].
One of the main methods for controlling outbreaks of bacterial diseases in fish farms is antibiotic
therapy [11]. However, there are already reports of Lactococcus spp. strains becoming resistant to the
main drugs used in aquaculture [12,13]. The indiscriminate use of antimicrobials has been reported
by producers and technicians from different fish farms, which can result in bacterial resistance to
specific drug [14]. As a result, a substance already used by a producer may no longer be effective in
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© 2024 by the author(s). Distributed under a Creative Commons CC BY license.
2
treating bacteriosis, thereby necessitating the use of another antibiotic. Additionally, it is worth
mentioning that the rate of approval for new drugs is slower than the evolution of bacterial resistance,
leading producers to use off-label drugs [15].
One way to monitor antimicrobial resistance in lactococcosis-causing bacteria is through the use
of laboratory methods such as disk diffusion [10] and broth dilution [16] methods. The former is
considered an inexpensive, reliable and simple technique that can be easily applied in a laboratory
routine, while in comparison the latter is technically demanding and labor-intensive [17]. However,
in Brazil, few studies have evaluated these methodologies for testing Lactococcus spp. strains, whether
using isolates from terrestrial mammals or aquatic animals. In Brazil, the disk diffusion assay has
been performed for isolates of L. petauri from farmed Oreochromis niloticus, and resistance for some
isolates to amoxicillin, erythromycin, florfenicol and norfloxacin was identified. In addition, all the
isolates evaluated were considered resistant to trimethoprim/sulfamethoxazole [10]. Bacteria of the
genus Lactococcus have also been isolated from native Brazilian fish species [2], and little is known
about the use of antimicrobials in these species and their antimicrobial susceptibility profiles.
The main problem in determining the sensitivity of bacterial fish pathogens to antimicrobials is
the lack of reference values. Without these values, it is not possible to determine whether an isolate
is sensitive or resistant. There are no internationally recognized epidemiological cut-off values for
disk diffusion data for Lactococcus spp. strains in the Clinical Laboratory Standards Institute (CLSI)
or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Previous
studies have generated provisional epidemiological cut-off values for L. petauri from disk diffusion
zone data using the normalized resistance interpretation (NRI) method [10] for L. garvieae and L.
petauri from minimum inhibitory concentration data using NRI and ECOFFinder approaches [16].
Nevertheless, for disk diffusion zone data, there are no reports of established cut-off values in the
literature for L. garvieae and L. formosensis.
Therefore, the aim of this study was to evaluate the susceptibility profile of Lactococcus spp.
strains obtained from native Brazilian fish species to different antimicrobials and to calculate the
provisional epidemiological cut-off values (pECVs) for Lactococcus garvieae.
2. Materials and Methods
2.1. Bacterial Strains and Identification
A total of 47 Lactococcus spp. strains (n = 6 L. formosensis, n = 17 L. garvieae and n = 24 L. petauri)
were used in this study. The isolates were obtained from eleven native fish species (Arapaima gigas,
Brycon amazonicus, Cichla sp., Colossoma macropomum, Hoplias macrophtalmus, Hoplias malabaricus,
Lophiosilurus alexandri, Phractocephalus hemioliopterus, Pseudoplatystoma corruscans, Pseudoplatystoma
fasciatum and a hybrid of Pseudoplatystoma) originating from free-living fish or commercial farms,
between 2012 and 2024, in six Brazilian states (Amazonas, Bahia, Mato Grosso do Sul, Minas Gerais,
Pará and São Paulo) (Table 1) [2,18–22]. These isolates were obtained through routine laboratory
diagnosis of bacterial diseases in fish conducted by the Laboratory of Aquatic Animal Diseases
(Veterinary School, Federal University of Minas Gerais, Belo Horizonte, Brazil), Laboratory of
Applied Microbiology of Aquatic Organisms (Nilton Lins University, Manaus, Brazil), Laboratory of
Microbiology and Parasitology of Aquatic Organisms (Aquaculture Center of São Paulo State
University, São Paulo, Brazil), and Fisheries Institute (São Paulo, Brazil). Of these, 11, originating
from Arapaima gigas (n = 7), Cichla sp. (n = 1), Hoplias malabaricus (n = 1) and Pseudoplatystoma sp. (n =
2), were recovered through bacterial examination after the recent disease outbreak. Furthermore, all
the selected isolates were identified to species level using matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Germany) [20] with the Bruker
MALDI Biotyper database (v13.0.0.2) followed by gyrB sequencing [23]. The isolates were stored at -
70 °C in BHI broth with 15% glycerol until use.
Table 1. Metadata of the 47 strains of lactococcosis-causing bacteria isolated from the native Brazilian
fish species.
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3
Isolate
Species
Host
Origin
Tissue
Year
State
Reference
167/23-02
L. formosensis
Arapaima gigas
Farmed
Brain
2023
BA
[2]
167/23-06
L. formosensis
Arapaima gigas
Farmed
Brain
2023
BA
[2]
167/23-09
L. formosensis
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
AM-LG05
L. formosensis
Colossoma macropomum
Farmed
Intestine
2022
AM
[2]
52MS
L. formosensis
Pseudoplatystoma fasciatum
Farmed
Brain
2012
MS
[18]
LG91-23
L. formosensis
Pseudoplatystoma sp.
Farmed
Brain
2023
MG
[2]
CRBP53
L. garvieae
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
CRBP54
L. garvieae
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
CRBP138
L. garvieae
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
CRBP144
L. garvieae
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
PA-LG01
L. garvieae
Arapaima gigas
Farmed
Brain
2018
PA
[22]
LG88-23
L. garvieae
Brycon amazonicus
Farmed
Brain
2023
MG
[2]
LG89-23
L. garvieae
Brycon amazonicus
Farmed
Kidney
2023
MG
[2]
LG116-23
L. garvieae
Cichla sp.
Wild
Brain
2023
MG
This
study
LG63-21
L. garvieae
Hoplias macrophtalmus
Farmed
Kidney
2021
MG
[2]
LG114-23
L. garvieae
Hoplias malabaricus
Wild
Brain
2023
AM
This
study
LG10-14
L. garvieae
Lophiosilurus alexandri
Farmed
Brain
2014
MG
[20]
LG66-22
L. garvieae
Phractocephalus hemioliopterus
Farmed
Kidney
2022
MG
[2]
LG09-14
L. garvieae
Pseudoplatystoma corruscans
Farmed
Kidney
2014
SP
[20]
LG23-16
L. garvieae
Pseudoplatystoma corruscans
Farmed
Brain
2016
SP
[21]
177
L. garvieae
Pseudoplatystoma fasciatum
Farmed
Brain
2012
MS
[19]
31MS
L. garvieae
Pseudoplatystoma fasciatum
Farmed
Kidney
2012
MS
[18]
LG119-24
L. garvieae
Pseudoplatystoma sp.
Farmed
Brain
2024
MG
This
study
167/23-03
L. petauri
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
167/23-04
L. petauri
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
167/23-05
L. petauri
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
167/23-07
L. petauri
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
167/23-08
L. petauri
Arapaima gigas
Farmed
Kidney
2023
BA
This
study
167/23-10
L. petauri
Arapaima gigas
Farmed
Spleen
2023
BA
This
study
CRBT89
L. petauri
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
CRBT98
L. petauri
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
CRBP146
L. petauri
Arapaima gigas
Farmed
Intestine
2023
AM
[2]
AM-LG07
L. petauri
Brycon amazonicus
Farmed
Brain
2022
AM
[2]
AM-LG08
L. petauri
Brycon amazonicus
Farmed
Brain
2022
AM
[2]
AM-LG02
L. petauri
Colossoma macropomum
Farmed
Intestine
2020
AM
[2]
AM-LG03
L. petauri
Colossoma macropomum
Farmed
Intestine
2022
AM
[2]
LG03-18
L. petauri
Pseudoplatystoma corruscans
Farmed
Brain
2018
MG
[2]
14MS
L. petauri
Pseudoplatystoma fasciatum
Farmed
Kidney
2012
MS
[18]
176
L. petauri
Pseudoplatystoma fasciatum
Farmed
Brain
2012
MS
[19]
86
L. petauri
Pseudoplatystoma sp.
Farmed
Brain
2012
MS
[19]
89/2
L. petauri
Pseudoplatystoma sp.
Farmed
Brain
2012
MS
[19]
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93
L. petauri
Pseudoplatystoma sp.
Farmed
Brain
2012
MS
[19]
LG86-23
L. petauri
Pseudoplatystoma sp.
Farmed
Kidney
2023
MG
[2]
LG94-23
L. petauri
Pseudoplatystoma sp.
Farmed
Brain
2023
MG
[2]
LG104-23
L. petauri
Pseudoplatystoma sp.
Farmed
Brain
2023
MG
[2]
LG106-23
L. petauri
Pseudoplatystoma sp.
Farmed
Kidney
2023
MG
[2]
LG117-23
L. petauri
Pseudoplatystoma sp.
Farmed
Kidney
2023
MG
This
study
AM: Amazonas; BA: Bahia; MS: Mato Grosso do Sul; MG: Minas Gerais; PA: Pará; SP: São Paulo.
2.2. Susceptibility Testing
Disk diffusion tests against Lactococcus spp. were carried out according to the protocol provided
in the CLSI guideline VET03, with adaptations recommended for bacteria of the genus Streptococcus
(Group 4) [24]. The disks used contained 10 µg amoxicillin, 15 µg erythromycin, 30 µg florfenicol, 10
µg neomycin, 10 µg norfloxacin, 30 µg oxytetracycline and 1.25/23.75 µg
trimethoprim/sulfamethoxazole. The disks were obtained from a commercial company (Oxoid,
United Kingdom).
The selected isolates (Table 1) were thawed, inoculated onto Man Rogosa & Sharpe (MRS, Merck,
Germany) agar, and incubated at 28 ºC for 48 h. After incubation, colonies were collected and
suspended in sterile saline solution until they reached an absorbance of between 0.08 and 0.13 (DO625)
nm using a spectrophotometer (Spectrum, China). Muller-Hinton agar enriched with 5% defibrinated
sheep blood was inoculated with the bacterial suspension using sterile swabs. Then, antimicrobial
disks were placed on the agar and the plates were incubated at 28 ºC for 24 h. Additionally, the quality
control reference strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida
ATCC 33658 were grown on blood agar, incubated at 28 ºC for 24 h and subjected to the same
experimental conditions described above as recommended by the CLSI for this method. All the
procedures were performed in duplicate. The diameter of the inhibition zone of all the isolates was
measured.
2.3. Calculation of Provisional Lactococcus garvieae Epidemiological Cut-Off Values
As there is no established zone diameter cut-off for Lactococcus garvieae generated by a standard
method, this study calculated the pECV for each antimicrobial agent tested using the automatic
normalized resistance interpretation (NRI) method (www.bioscand.se/nri) from the inhibition zone
data that was generated [25,26]. The isolates were then classified as wild-type (WT) or non-wild-type
(NWT) [27]. To meet the minimum requirements of the NRI method [28], the disk diffusion data of
the Lactococcus garvieae strains isolated from Oreochromis niloticus (n = 3), Trichogaster lalius (n = 1), and
Xiphophorus maculatus (n = 1) from the Laboratory of Aquatic Animal Diseases culture collection were
included in the calculation of the pECVs (Supplementary Table 2).
2.4. Data Analysis
Lactococcus petauri strains were classified as WT or NWT according to the previously established
pECV (Egger et al. 2023). However, as Lactococcus formosensis has no number of suggested
observations to set a reliable pECV calculation, the inhibition zone data were shown as maximum,
minimum, mean and standard deviation values. Regardless of the bacterial species, bacteria that did
not present an inhibition zone and did not have a defined ECV were considered NWT for the
antimicrobials. R software v.4.3.1 [29] and RAWGraphs v2.0 [30] were used for data visualization.
Isolates classified as NWT for at least three classes of antimicrobials were considered to be multidrug-
resistant bacteria [31]. The multiple antibiotic resistance (MAR) index was also calculated [32].
3. Results
3.1. Bacterial Identification
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From the sequencing of the gyrB gene, Lactococcus formosensis (n = 1, Arapaima gigas), L.
garvieae (n = 1, Cichla sp.; n = 1, Hoplias malabaricus; n = 1, Pseudoplatystoma sp.) and L. petauri (n
= 6, Arapaima gigas; n = 1, Pseudoplatystoma sp.) were detected from the current disease outbreaks
in native fish species (Table 1). The gyrB gene sequences of these isolates were included in the NCBI
database.
The remaining isolates used in this study were identified at the species level in a previous study
[2].
3.2. Quality Control
The reference strains E. coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC
33658 presented inhibition zone diameters within the acceptable ranges established by the CLSI
(Table 2).
Table 2. Minimum and maximum values, mean and standard deviation of the inhibition zone
diameters, epidemiological cut-off values, and wild type/non-wild type (WT/NWT) percentual for
Lactococcus spp. and quality control strains in the antimicrobial susceptibility analysis.
Antimicrobials
Minimum
Value
Maximum
Value
Mean
± SD
ECV
(mm)
WT
(%)
NWT*
(%)
Lactococcus formosensisa
Amoxicillin
19
27
23.2 ±
2.7
-
-
-
Erythromycin
20
30
25.7 ±
3.7
-
-
-
Florfenicol
6
28
22.3 ±
7.8
-
-
16.7
Neomycin
10
17
14.9 ±
2.4
-
-
-
Norfloxacin
6
6
6.0 ±
0.0
-
-
100
Oxytetracycline
6
27
9.5 ±
7.5
-
-
66.7
Trimethoprim-
sulfametoxazole
6
6
6.0 ±
0.0
-
-
100
Lactococcus garvieaeb
Amoxicillin
18
28
21.4 ±
2.2
≥ 11
100
0
Erythromycin
16
31
24.7 ±
3.7
≥ 16
100
0
Florfenicol
6
29
20.9 ±
4.4
≥ 12
94.4
5.6
Neomycin
10
19
15.1 ±
2.7
≥ 7
100
0
Norfloxacin
6
19
9.0 ±
4.0
-
-
47
Oxytetracycline
6
27
16.5 ±
7.3
≥ 10
72.2
27.8
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Trimethoprim-
sulfametoxazole
6
19
7.2 ±
3.4
-
-
88.2
Lactococcus petauric
Amoxicillin
15
26
20.5 ±
3.0
≥ 16
95.8
4.2
Erythromycin
6
31
22.4 ±
7.1
≥ 23
66.7
33.3
Florfenicol
6
29
19.5 ±
6.9
≥ 21
62.5
37.5
Neomycin
10
19
14.2 ±
2.2
≥ 9
100
0
Norfloxacin
6
14
8.6 ±
2.9
≥ 13
16.7
83.3
Oxytetracycline
6
26
13.6 ±
7.5
≥ 23
16.7
83.3
Trimethoprim-
sulfametoxazole
6
14
6.3 ±
1.4
-
-
95.8
Escherichia coli ATCC 25922d
Amoxicillin
14
19
15.8 ±
2.4
-
-
-
Erythromycin
12
18
14.6 ±
2.8
-
-
-
Florfenicol
19
28
23.5 ±
4.4
-
-
-
Neomycin
16
20
18.0 ±
2.0
-
-
-
Norfloxacin
24
34
30.6 ±
5.7
-
-
-
Oxytetracycline
19
27
23.2 ±
3.3
-
-
-
Trimethoprim-
sulfametoxazole
25
26
25.5 ±
0.7
-
-
-
Aeromonas salmonicida subsp. salmonicida ATCC 33658d
Amoxicillin
24
30
27.4 ±
3.1
-
-
-
Erythromycin
19
22
20.7 ±
1.5
-
-
-
Florfenicol
32
36
34.2 ±
1.7
-
-
-
Neomycin
12
20
17.3 ±
4.6
-
-
-
Norfloxacin
21
37
29.6 ±
8.0
-
-
-
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Oxytetracycline
29
32
29.7 ±
1.5
-
-
-
Trimethoprim-
sulfametoxazole
24
26
25.0 ±
1.4
-
-
-
a ECV undetermined b pECV determined in this study c pECV determined in Egger et al. [10] d Quality control
strains * Regardless of the bacterial species, bacteria that did not present an inhibition zone and did not have a
defined epidemiological cutoff value (ECV) were considered NWT for the antimicrobials.
3.3. Antimicrobial Susceptibility for Lactococcus formosensis
The disk diffusion assay for L. formosensis strains exhibited zones ranging between 6 mm and 30
mm (Table 2 and Supplementary Table 1). All the isolates were categorized as NWT (no observation
of inhibition zone) for trimethoprim/sulfamethoxazole and norfloxacin (Figure 1). A total of one and
four strains were categorized as NWT for florfenicol and oxytetracycline, respectively (Figure 1). For
these antimicrobials, all the isolates from Arapaima gigas were categorized as NWT for
oxytetracycline, and the LG91-23 strain (from Pseudoplatystoma sp.) was NWT for both drugs (Figure
2). Since there is no pECV for Lactococcus formosensis, it is not possible to determine whether the other
isolates are resistant to other antimicrobials. In addition, four isolates were classified as multidrug-
resistant. The MAR index of the isolates varied between 0.285 and 0.571 (Figure 3, Supplementary
Table 1).
Figure 1. Disk diffusion scatter plots for antimicrobials versus diameters of inhibition zones for the
six Lactococcus formosensis, 17 Lactococcus garvieae and 24 Lactococcus petauri strains evaluated.
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Figure 2. Alluvial plot demonstrating the association of the host with antimicrobial susceptibility or
resistance to florfenicol (FLO) and oxytetracycline (OXY) in Lactococcus formosensis (a), Lactococcus
garvieae (b) and Lactococcus petauri (c) strains.
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Figure 3. Multiple antibiotic resistance (MAR) index box plot of Lactococcus spp. strains isolated from
native Brazilian fish species. .
3.4. Antimicrobial Susceptibility for Lactococcus garvieae
The disk diffusion assay for L. garvieae strains exhibited zones raging between 6 mm and 31 mm
(Table 2 and Supplementary Table 1). The distribution of the inhibition zones obtained from all the
evaluated isolates are shown in Supplementary Figure 1. The calculated pECVs for the antimicrobials
are presented in Table 2. A total of eight and fifteen isolates (Figure 1) presented a zone of complete
inhibition of 6 mm for norfloxacin and trimethoprim/sulfametoxazole, respectively, preventing the
establishment of the ECV for these antimicrobials. However, these isolates were categorized as NWT.
Based on the calculated pECVs, all the isolates were classified as WT for amoxicillin, erythromycin
and neomycin. One and five isolates were classified as NWT for florfenicol and oxytetracycline,
respectively, especially those strains isolated from Pseudoplatystoma sp. (Figure 2). In addition, two
isolates were classified as multidrug-resistant. The MAR index of the isolates varied between 0.00
and 0.428 (Figure 3, Supplementary Table 1).
3.5. Antimicrobial Susceptibility for Lactococcus petauri
The disk diffusion assay for the L. petauri strains exhibited zones ranging between 6 mm and 31
mm (Table 2 and Supplementary Table 1). All the isolates were classified as WT for neomycin. A total
of 1, 8, 11, 22, 22 and 23 isolates were classified as NWT for amoxicillin, erythromycin, florfenicol,
oxytetracycline, norfloxacin and trimethoprim/sulfametoxazole, respectively (Supplementary Table
1). A resistance phenotype for florfenicol and oxytetracycline was observed in all the native Brazilian
fish species in which L. petauri was isolated (Figure 2). Twenty-one isolates were classified as
multidrug-resistant. The MAR index varied between 0.285 and 0.857 (Figure 3, Supplementary Table
1).
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4. Discussion
Currently, antimicrobial resistance is one of the biggest threats to public health [33], especially
with the emergence of multidrug-resistant strains [34]. The antimicrobial susceptibility profile in
lactococcosis-causing bacteria strains has been studied by several different institutions and
researchers using various techniques, such as disk diffusion [35,36], broth dilution [12,37] and the
Etest [38]. Studies have shown that most of the isolates evaluated are resistant to ampicillin [36,39],
florfenicol [40], flumequine [35], nalidixic acid [39,41,42], norfloxacin [40], tetracycline [13] and
trimethoprim/sulfamethoxazole [35,39,41,43]. Although detected at lower percentages, there are also
records of resistance to amoxicillin (16-23%), bacitracin (42%), ciprofloxacin (4%), chloramphenicol
(18%), enrofloxacin (33-67%), erythromycin (16-52%), kanamycin (33%), oxytetracycline (4-44%) and
streptomycin (33%) [13,35,36,39,42,43].
In the scientific literature, it is possible to observe heterogeneity in the antimicrobial resistance
profiles for Lactococcus spp. strains, which may be related to the different species within the genus.
This is because most of the articles, including some recent ones, did not perform the correct taxonomic
classification of the isolates, which is currently recommended [44]. Only three studies assessed the
antimicrobial resistance profile after correct species identification, using the disk diffusion [10,45] and
broth dilution [16]. Furthermore, Öztürk et al. [16] suggest that this heterogeneity is related to the
overuse or misuse of antimicrobials at the farm level and the lack of established susceptibility cut-off
values for each of the three species that cause piscine lactococcosis. Here, we evaluated the
antimicrobial resistance profiles of L. formosensis, L. garvieae and L. petauri strains isolated from native
fish species in Brazil using disk diffusion susceptibility testing and established pECVs for five out of
seven antimicrobials for L. garvieae strains.
Regardless of the bacterial species evaluated in our study, the trimethoprim/sulfametoxazole
resistance phenotype stood out (L. formosensis = 100%, L. garvieae = 88.2%, L. petauri = 95.8%) (Figure
1). Resistance to this drug has previously been reported in the literature for Lactococcus spp. strains
isolated from Oncorhynchus mykiss, Dicentrarchus labrax and Oreochromis niloticus [6,10,35,39–41,43].
For the other drugs, interspecies variation has been observed.
Unfortunately, due to the limited number of isolates identified as L. formosensis in our study, it
was not possible to establish a pECV and, therefore, classification as WT or NWT could not be
performed. However, we considered those isolates for which no inhibition zones were observed for
the antimicrobials tested to be NWT. Thus, in addition to trimethoprim/sulfametoxazole, all the
isolates were considered NWT for norfloxacin. This result is in disagreement with the study
conducted by Lin et al. [46], in which all the L. formosensis strains isolated from milk samples of a cow
with clinical mastitis were susceptible to quinolones via broth dilution testing. We also did not
observe the formation of inhibition zones in four isolates (three from Arapaima gigas and one from
Pseudoplatystoma sp.) for oxytetracycline, nor in one Pseudoplatystoma sp. isolate (LG91-23) for
florfenicol (Figure 2a). Chan et al. [45] evaluated susceptibility using the disk diffusion method for
an L. formosensis strain obtained from a human with bacteremia and found that the isolate was
susceptible to tetracycline. There is no mention in the literature regarding resistance profiles to
florfenicol, regardless of the host evaluated; however, a previous study demonstrated resistance to
another amphenicol, chloramphenicol, for all the isolates evaluated [46]. Although we cannot
determine susceptibility for other drug classes, the literature mentions L. formosensis resistance to
aminoglycosides and macrolides, and susceptibility to β-lactams [45,46]. If we consider the pECV of
L. garvieae and L. petauri from this study, all the isolates would be classified as WT for amoxicillin,
erythromycin, florfenicol and neomycin, which would corroborate the previous information.
Additionally, the AM-LG05 strain would be classified as NWT for oxytetracycline, increasing the
number of multidrug-resistant isolates. It was possible to observe that the antimicrobial resistance
profile was similar among the Arapaima gigas isolates, as all the isolates share the same geographic
origin.
For the L. petauri strains, we compared the results using the previously established pECV. All
the isolates evaluated were classified as WT for neomycin, thereby corroborating with Egger et al.
[10]. For amoxicillin and erythromycin, our isolates demonstrated a low frequency of NWT detection,
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4.1% and 33.3%, respectively, when compared to other antimicrobials. A previous study
demonstrated resistance of 6% and 25% for L. petauri strains isolated from Oncorhynchus mykiss for
erythromycin and amoxicillin, respectively [16]. For isolates obtained from Oreochromis niloticus, the
NWT percentages were lower, around 3% for both antimicrobials [10]. A high percentage of isolates
classified as NWT for norfloxacin was observed in our study (91.7%) compared to the results obtained
in Oreochromis niloticus (16.75%) [10]. Regarding florfenicol and oxytetracycline, 11.4% and 91.6% of
the isolates were classified as NWT, respectively. In Oncorhynchus mykiss and Oreochromis niloticus
isolates, NWT values of 0% and 12.5% for oxytetracycline and 3.4% to 9.4% for florfenicol have been
observed [10,16]. The resistance profile was similar among the Pseudoplatystoma sp. isolates from the
states of Mato Grosso and Minas Gerais, as well as most of the Arapaima gigas isolates from Bahia
(Supplementary Table 1). However, for isolates obtained from this latter fish species in the northern
region of Brazil, individual variation was detected, as were the cases with Colossoma macropomum and
Brycon amazonicus isolates (Figure 2c).
There are no studies that have standardized cut-off values, even provisional ones, for L. garvieae
strains following its correct taxonomic identification. Therefore, our study is the first to do so.
However, we emphasize that the ECVs presented here are provisional. To generate ECVs that are
relevant to disk diffusion data for L. garvieae, a larger number of isolates (over 100 observations) from
at least five different laboratories would be required [28]. As previously mentioned, all the isolates
evaluated were classified as being WT for amoxicillin, erythromycin and neomycin. In contrast,
isolates from Oncorhynchus mykiss exhibited varying susceptibility to these antimicrobials.
Approximately 2.6%, 24.4% and 6.4-11.5% of the isolates were resistant to amoxicillin, erythromycin
and aminoglycosides, respectively [16]. A total of 47% of the isolates from native fish species in Brazil
were considered to be NWT for norfloxacin, in contrast to previous studies that reported low (7.7%)
or no resistance to quinolones [16,45]. A total of 5.8% and 29.4% of the isolates were classified as NWT
for florfenicol and oxytetracycline, respectively. However, the literature reports resistance rates of
26.9% for florfenicol and 17.9% for oxytetracycline [16]. The antimicrobial resistance profile observed
in our study for the L. garvieae strains was not consistent among the aquatic host species analyzed or
with the origin of the isolates, demonstrating a heterogeneous profile (Figure 2b).
Our study showed that the L. garvieae strains tend to be more sensitive to antimicrobials when
compared to the L. formosensis and L. petauri strains. Furthermore, the proportion of L. garvieae isolates
with a MAR index greater than 0.3 (11.7%) was lower than that found in L. formosensis (66.7%) and L.
petauri (87.5%) (Figure 3). The most efficient measure to control bacterial diseases is the use of
antimicrobials [11]. However, since the isolates evaluated in this study were classified as multi-
resistant to several antimicrobials (Supplementary Table 1), treating piscine lactococcosis in native
Brazilian fish species becomes challenging. Unfortunately, little is known about the use of
antimicrobials in native fish species in Brazil. However, prophylactic and metaphylactic use of
antimicrobials, especially oxytetracycline, in larviculture of native species and during the feeding
training of carnivorous species like Pseudoplatystoma sp. and Arapaima gigas has been reported by
producers and technicians in the country. Additionally, it is known that commercial fish farming in
Brazil involves off-label use of amoxicillin, enrofloxacin and norfloxacin [15,47]. In the central-
western region of Brazil, fluoroquinolones, especially norfloxacin and enrofloxacin, intended for the
treatment of cattle, have also been used off-label in Pseudoplatystoma sp.. Therefore, the widespread
use of these antimicrobials may have contributed to the increase in resistance among Lactococcus spp.
strains. It is also worth mentioning that when MAR index values exceed 0.2 (in our case, over 0.3 due
to the number of antibiotics tested), a high environmental risk of spreading antimicrobial resistance
is predicted [32]. In this context, the shared production of native fish species and Oreochromis niloticus
could pose a risk of transmitting antimicrobial-resistant Lactococcus spp. strains, or it could enable L.
petauri isolates from Oreochromis niloticus to acquire resistance genes in this production environment,
resulting in an unsatisfactory therapeutic approach during disease outbreaks. Therefore, monitoring
of antimicrobial resistance in Lactococcus spp. strains becomes essential.
Sun et al. [48] report that acquired resistance in microorganisms occurs for two reasons: the
natural resistance of bacteria to certain antimicrobials and acquired resistance due to continuous
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exposure to antimicrobials. Once the bacteria becomes resistant, this resistance can be transferred to
the other bacterial species through horizontal gene transfer [49]. Furthermore, some L. garvieae strains
carry these antimicrobial resistance genes on transferable R plasmids [35]. The acquisition and
transfer of antimicrobial resistance genes have been considered to be responsible for the spread and
distribution of antimicrobial resistance [16]. Previous studies indicate a high prevalence of
antimicrobial resistance genes in Lactococcus spp. isolates from Oncorhynchus mykiss [13,16,35]. There
is no description of the detection of resistance genes in Lactococcus spp. strains from Brazilian fishes.
However, given the higher percentage of multi-resistant isolates, future studies should be conducted
to identify resistance genes, particularly those encoding antimicrobial resistance, using genomic
tools.
In Brazil, only florfenicol and oxytetracycline are approved antimicrobial agents for use in
aquaculture [50]. Both antimicrobials act against Gram-negative and Gram-positive bacteria; they are
bacteriostatic drugs that work by binding to bacterial ribosomal subunits and inhibiting protein
synthesis [49]. However, neither of these antimicrobials has been evaluated for their therapeutic
efficacy in fish either naturally or experimentally infected with Lactococcus spp. in Brazil.
Nevertheless, the administration of oxytetracycline in Oncorhynchus mykiss in Greece was reported to
be unsatisfactory in both prophylactic and therapeutic treatments [51]. The circulation of florfenicol-
and oxytetracycline-resistant strains in Brazilian fish farms could become a significant health issue
when producing native species. The oral administration of amoxicillin, erythromycin and flumequine
did not yield significant results in the treatment of Oncorhynchus mykiss and Dicentrarchus labrax with
lactococcosis [6,51]. However, based on the antimicrobial susceptibility tests from our study,
amoxicillin and neomycin could be tested for their therapeutic efficacy against piscine lactococcosis
in Brazil.
5. Conclusions
When the pECVs of L. garvieae strains and the antimicrobial resistance profiles of L. garvieae, L.
formosensis and L. petauri were assessed, a higher percentage of resistance to various antimicrobials
was observed among the evaluated isolates, especially for L. petauri, including multidrug-resistant
strains. This is quite different from what has been observed in Oreochromis niloticus farms in Brazil,
thus making it essential to monitor the susceptibility of the isolates and raise awareness among
producers about the correct use of antibiotics.
Supplementary Materials: The following supporting information can be downloaded at the website of this
paper posted on Preprints.org, Figure S1: Histograms of the inhibition zone for Lactococcus garvieae strains
against amoxicillin (AMO), erythromycin (ERY), florfenicol (FLO), neomycin (NEO), norfloxacin (NOR),
oxytetracycline (OXY) and trimethoprim/sulfamethoxazole (SXT); Table S1: Inhibition zones diameters (mm) of
antimicrobial agents against Lactococcus spp. strains determined using disk diffusion susceptibility assay and
MAR index calculated per isolate; Table S2: Inhibition zones diameters (mm) of antimicrobial agents against
Lactococcus garvieae strains used to satisfy the minimum requirements of the NRI method.
Author Contributions: Conceptualization: AECR, ACCB, HCM, HCPF and GCT; Methodology: AECR, ACCB,
HCM, CRMSM, JMT, HCPF and GCT; Formal analysis: AECR, ACCB and HCM; Investigation: AECR, ACCB,
HCM, CRMSM, LFFN, FP, SUG, CAGL, HCPF and GCT; Resources: FP, SUG, CAGL, HCPF and GCT; Data
curation: LFFN and GCT; Writing—original draft preparation, AECR and GCT; Writing—review and editing,
AECR, ACCB, HCM, CRMSM, JMT, LFFN, FP, SUG, CAGL, HCPF and GCT; Visualization: GCT; Supervision:
SUG, HCPF and GCT; Project administration: GCT; Funding acquisition: HCPF and GCT. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
– Brasil (CAPES) through the PROCAD/Amazônia (grant number 88881.200614/2018-01) and PDPG-
CAPES, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, grant numbers APQ-01227-22,
APQ-04309-22 and PPM-00779-18), and Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM,
grant number 01.02.016301.03071/2022-11).
Ethical Approval: No ethical approval was required as the research in this article is related to microorganisms.
Data Availability Statement: The gyrB gene sequences of the Lactococcus spp. strains isolated from the native
Brazilian fish species were included in the NCBI database as follows: L. formosensis – 167/23-09: PQ529765; L.
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 October 2024 doi:10.20944/preprints202410.2381.v1
13
garvieae – LG114-23: PQ529769, LG116-23: PQ529771, LG119-24: PQ529773; L. petauri – 167/23-03: PQ529760,
167/23-04: PQ529761, 167/23-05: PQ529762, 167/23-07: PQ529763, 167/23-08: PQ529764, 167/23-10: PQ529766,
LG117-23: PQ529772.
Acknowledgments: The authors gratefully acknowledge the support provided by Maria José Tavares Ranzani-
Paiva for conducting this study.
Conflicts of Interest: The authors declare that there are no conflicts of interest.
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