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Journal of Fish Diseases 2015 doi:10.1111/jfd.

12337

Short Communication

Persistence of a single clone of Streptococcus agalactiae


causing disease in tilapia (Oreochromis sp.) cultured in
Colombia over 8 years

P Barato1, E R Martins2, J Melo-Cristino2, C A Iregui1 and M Ramirez2


1 Veterinary Pathobiology Research Group, Laboratory of Veterinary Pathology, Institute of Biotechnology, Universi-
dad Nacional de Colombia, Bogot!a, Colombia
2 Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,
Lisboa, Portugal

Keywords: Colombia, molecular epidemiology, Streptococcus agalactiae affects a large range of


Streptococcus agalactiae, tilapia. hosts which include humans, cows, dogs, cats, sea
mammals, crocodiles, bullfrogs and fish (Delannoy
Streptococcosis is a disease with major health and et al. 2013). While three capsular types (Ia, Ib
economic impacts on the tilapia industry world- and III) and a particular clonal complex (CC552)
wide (Mian et al. 2009). In 1997, more than US have been associated with strains isolated from fish
$150 million of annual losses representing treat- species, other sequence types have been found in
ment, morbidity and mortality were recorded and strains recovered from fish, marine mammals and
this value increased to US$250 million by 2008 humans (Evans et al. 2008; Delannoy et al. 2013;
(Klesius, Shoemaker & Evans 2008). In Colom- Godoy et al. 2013). Some S. agalactiae strains
bia, Streptococcus agalactiae (Lancefield group B from Kuwait share the same sequence type (ST7)
streptococci; GBS) is the main bacterial pathogen and capsular type (Ia) of human isolates from
of tilapia (Jim!enez et al. 2007), causing heavy but Japan (Evans et al. 2008). Both ST283 and
not yet quantified losses. ST491 were detected in fish from South-East Asia
Classical capsular serotyping and antimicrobial and were found to share the three-set genotype of
susceptibility testing have been extensively used the emerging ST283 clone associated with invasive
for characterizing GBS. More recently, DNA- disease in human adults in Asia (Delannoy et al.
based typing methods including pulsed-field gel 2013). ST103 was isolated from a pregnant
electrophoretic profiling (PFGE) and multilocus woman from Canada in 1999 (Springman et al.
sequence typing (MLST) have been increasingly 2009), and recently associated with diseased tilapia
used for typing GBS isolates. These techniques in Brazil (Godoy et al. 2013).
have been helpful in discriminating GBS popu- It has been proposed that fish consumption
lations into genetic lineages, with specific clones may increase the risk of acquiring GBS expressing
recognized by their enhanced virulence potential, capsular types Ia and Ib but not of other capsular
specific disease syndromes or tropism for partic- types (Foxman et al. 2007). In addition, Pereira
ular hosts (Jones et al. 2003; Martins et al. et al. (2010) demonstrated that some S. agalactiae
2007). strains of human and bovine origin can infect fish
and that genetic similarity between strains is not a
Correspondence M Ramirez, Instituto de Microbiologia, Facul- prerequisite for this to occur.
dade de Medicina, Universidade de Lisboa, Av. Professor
Egas Moniz, 1649-028 Lisboa, Portugal, Tel:+351 217 999 460 These studies have raised the possibility that
(e-mail: ramirez@fm.ul.pt) limited interspecies exchange may occur between

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Journal of Fish Diseases 2015 P Barato et al. GBS in tilapia in Colombia

humans and fish (Evans et al. 2008; Delannoy Identification of the GBS alpha-like protein genes
et al. 2013; Godoy et al. 2013), as was previously (bca, alp2/3, alp4, rib and epsilon) was done by
suggested between humans and their livestock, multiplex PCR (Creti et al. 2004). An additional
further strengthening the possible zoonotic origin multiplex PCR was performed for the identifica-
of some GBS infections (Oliveira et al. 2006; tion of pilus islands 1 and 2 (PI-1 and PI-2), the
Manning et al. 2010). later presenting two distinct variants, PI-2a and
The aim of this study was to characterize PI-2b (Martins, Melo-Cristino & Ramirez 2010).
S. agalactiae isolates recovered from tilapia with PI-1-negative isolates were confirmed not to carry
streptococcosis and from the environment, during the pilus island or parts of it with an additional
an eight-year period in two States of Colombia, set of primers (Martins et al. 2010).
using serotyping, surface protein gene profiling, All isolates were subjected to PFGE as
pilus-associated gene profiling, PFGE and MLST. previously described (Martins et al. 2007). The
Twenty-four GBS isolates representing 6 inde- Bionumerics software (Applied Maths, Sint-Mar-
pendent disease events were recovered from dis- tens-Latem, Belgium) was used to generate a den-
eased tilapias between 2003 and 2011 (Table 1). drogram for comparison of the PFGE patterns.
All tilapias had been diagnosed with streptococco- MLST was performed by amplifying and sequenc-
sis by histopathology and microbiology methods ing seven housekeeping genes as previously
(Hernandez et al. 2009). Identification of the iso- described (Jones et al. 2003), and sequence type
lates to the species level was done by Gram stain, (ST) assignment performed by comparison with
colony morphology, catalase test, haemolytic prop- the S. agalactiae MLST database (http://pubmlst.
erties and a commercial latex agglutination tech- org/sagalactiae). Analysis of DNA sequences was
nique (Slidex Strepto B; bioM!erieux, Marcy done using the Bionumerics software (Applied
!
L’Etoile, France). Maths, Sint-Martens-Latem, Belgium).
Capsular serotyping of all isolates was carried All isolates presented non-haemolytic and very
out by a latex agglutination assay with a GBS se- mucoid colonies in sheep blood agar, and all were
rotyping commercial kit (Essum, Ume" a, Sweden) confirmed as Lancefield group B streptococci sero-
according to the manufacturer’s instructions. type Ib. None of the isolates amplified any of the

Table 1 Streptococcus agalactiae isolates from diseased tilapia

Isolation

Epidemiological event State Year Source No. of isolates Isolate

1 Tolima 2003 Brain 1 STiTa 03-01


Eye 1 STiTa 03-02
2 Huila 2005 Brain 1 STiBe 100
3 Tolima 2006 Brain 1 STiTo 06-59
4 Huila 2008 Kidney 1 SaTiBe 08-18
5 Huila 2009 Several internal organs 12 SaTiBe 09-16
SaTiBe 09-30
SaTiBe 09-32
SaTiBe 09-33
SaTiBe 09-34
SaTiBe 09-35
SaTiBe 09-37
SaTiBe 09-38
SaTiBe 09-41
S-57
S-58
S-67
6 Huila 2011 Brain 1 SaTiBe 1063-112C
Brain 1 SaTiBe 1063-124C*
Brain 1 SaTiBe 1063-129C
Brain 1 SaTiBe 1063-138C
Kidney 1 SaTiBe 1063-120R
Kidney 1 SaTiBe 1063-124R*
Kidney 1 SaTiBe 1063-138R

*Recovered from the same fish.

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Journal of Fish Diseases 2015 P Barato et al. GBS in tilapia in Colombia

genes encoding surface proteins (bca, rib, epsilon, recovered in Huila in 2008 had been evaluated
alp2/3 and alp4). All isolates presented the PI-2b for bac, IS1548 (hylB) and ISSag1 and ISSag2
pilus island. (lmb) by Delannoy et al. (2013), with the same
Two very similar PFGE profiles were identified. negative results. Amplification of the genes encod-
Twenty-three isolates showed the same PFGE pat- ing alpha-like proteins was also negative in all
tern, and one (isolated in Tolima) had 95% simi- strains. These findings are in agreement with the
larity with the remaining strains (Fig. 1). The absence of those genes in other CC552 GBS
PFGE profiles of the 24 GBS tilapia isolates were strains recovered from tilapia, possibly partly
compared with a database including 899 isolates reflecting species-specificity in the interactions
recovered from human colonization and infections between surface proteins and the infected host
(Martins et al. 2013) and also with 14 bovine iso- (Delannoy et al. 2013). Additionally, these results
lates (Trigo et al. 2008). Tilapia isolates clustered suggest that the S. agalactiae found in tilapia cul-
in a distinct group, sharing only 53% similarity tured in Colombia do not have a close genetic
with the closest human or bovine profiles (data relationship to any S. agalactiae found in humans.
not shown). All isolates were representatives of A combination of typing methods showed that
ST260 by MLST. only one clone of S. agalactiae has been circulat-
Our study characterized a collection of GBS ing in several regions of tilapia production in
isolates recovered from tilapia in Colombia cover- Colombia, including the main producing site,
ing different sites and years. The multiplex PCR Betania dam in Huila (23.991 ton/per year)
analysis indicated that all 24 isolates were negative (Merino, Bonilla & Bages 2013). All isolates rep-
for bca amplification, a finding similar to that resenting this clone were serotype Ib, ST260, had
reported by Godoy et al. (2013). The isolate pili 2b and did not carry genes encoding alpha or

Strain Serotype ST Alp Pilus


100
99
98
97
96

S-57 Ib 260 none PI-2b


S-58 Ib 260 none PI-2b
S-67 Ib 260 none PI-2b
SaTiBe 08-18 Ib 260 none PI-2b
SaTiBe 09-16 Ib 260 none PI-2b
SaTiBe 09-30 Ib 260 none PI-2b
SaTiBe 09-32 Ib 260 none PI-2b
SaTiBe 09-33 Ib 260 none PI-2b
SaTiBe 09-34 Ib 260 none PI-2b
SaTiBe 09-35 Ib 260 none PI-2b
SaTiBe 09-37 Ib 260 none PI-2b
SaTiBe 09-38 Ib 260 none PI-2b
SaTiBe 09-41 Ib 260 none PI-2b
SaTiBe 1063-112C Ib 260 none PI-2b
SaTiBe 1063-120R Ib 260 none PI-2b
SaTiBe 1063-124C Ib 260 none PI-2b
SaTiBe 1063-124R Ib 260 none PI-2b
SaTiBe 1063-129C Ib 260 none PI-2b
SaTiBe 1063-138C Ib 260 none PI-2b
SaTiBe 1063-138R Ib 260 none PI-2b
STiBe 100 Ib 260 none PI-2b
STiTa 03-01 Ib 260 none PI-2b
STiTa 03-02 Ib 260 none PI-2b
STiTo 06-59 Ib 260 none PI-2b

Figure 1 Dendrogram of the PFGE profiles of 24 GBS isolates. The dendrogram was constructed using the UPGMA method. Dice
coefficients (percentages) are indicated in the scale above the dendrogram. Serotype, sequence type (ST). Alpha and alpha-like pro-
tein (Alp) gene and pilus island profiling are indicated for each strain.

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Journal of Fish Diseases 2015 P Barato et al. GBS in tilapia in Colombia

alpha-like surface proteins, regardless of isolation In conclusion, this study reports the endemicity
year or origin. ST260 is part of clonal complex of a single clone of S. agalactiae in tilapia farms
CC552. This clonal complex consists of five STs in Colombia for 8 years, where it is responsible
exclusively found in fish isolates and includes iso- for significant economic losses to the fish industry.
lates recovered from the USA, Honduras, Colom- The development of control and prevention strate-
bia, Costa Rica and Brazil (Evans et al. 2008; gies could be based on the described characteristics
Delannoy et al. 2013; Godoy et al. 2013). Our of this clone. Future epidemiological work will be
data provide important additional information necessary to determine whether there is ongoing
supporting the association between this ST and transmission between multiple sites, potentially
fish farms in several countries of the northern key information to control streptococcosis in the
region of South America. Most of the isolates country.
(23/24) in our collection showed undistinguish-
able PFGE profiles. PFGE is believed to be a
Conflict of interest
highly discriminatory typing method (Carric!o
et al. 2013), and these data strongly argue that a Dr. Jos!e Melo-Cristino has received research
stable genetic lineage has been endemic in the grants administered through his university and
country in the last 8 years. received honoraria for serving on the speakers
The finding that a single clone of S. agalactiae bureaus of Pfizer, Bial, GlaxoSmithKline and
was present in the tilapia cultured in Colombia Novartis. Dr. M!ario Ramirez has received hono-
could suggest that it originates in the same source raria for serving on speakers bureau of Pfizer and
and is re-circulated in the fish culture system for consulting from GlaxoSmithKline. The other
(Amal et al. 2013b). In Colombia, the commercial authors declare no conflict of interest. No com-
trade of fingerlings between Huila and Tolima has pany or financing body had any interference in
been maintained during the past 10 years. We the decision to publish.
could not determine whether GBS is transported
with the water or with the animals moving
Acknowledgements
between these locations. Transportation of carrier
fingerlings or water contaminated with the patho- P.B. was supported by a grant from COLCIEN-
gen between tilapia farms with streptococcosis has CIAS, Scholarship to doctoral studies ‘Generaci!on
been described as the origin of S. agalactiae infec- Bicentenario’ No. 121731 and DIB grant to
tion in Malaysia (Amal et al. 2013a) and could research project No. 14357. E.R.M. was sup-
also be occurring in Colombia. ported by a grant from Fundac!~ao para a Ci^encia e
The presence of a single clone of GBS in fish in a Tecnologia (SFRH/BPD/80038/2011).
Colombia could be an advantage to establish pre-
vention strategies. Virulence factors, as the poly-
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infants and pregnant women in Portugal revealing two Accepted: 11 November 2014

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John Wiley & Sons Ltd 5

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