The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques.
The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques.
A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP-PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP-PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.
Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.
Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.
Streptococcus agalactiae is an important bacterial fish pathogen in the global aquaculture industry (Klesius et al. 2008). Infection caused by Strep. agalactiae had been reported in several species of marine and freshwater fishes, such as silver pomfret (Pampus argenteus), golden pompano (Trachinotus blochii), seabream (Sparus auratus), wild mullet (Liza klunzingeri), Nile tilapia (Oreochromis niloticus), red tilapia (Oreochromis sp.), ya-fish (Schizothorax prenanti), wild giant Queensland grouper (Epinephelus lanceolatus), estuary ray (Dasyatis fluviorum), mangrove whipray (Himantura granulata) and eastern shovelnose ray (Aptychotrema rostrata) (Evans et al. 2002; Duremdez et al. 2004; Suanyuk et al. 2005, 2008; Hernández et al. 2009; Mian et al. 2009; Geng et al. 2011; Amal et al. 2012; Azad et al. 2012; Bowater et al. 2012). In fish, Strep. agalactiae causes septicaemia, leading to high prevalence of meningoencephalitis, myocarditis and choroiditis (Evans et al. 2006). This bacterium is also associated with diseases in humans, dogs, cows, horses, dolphins and guinea pigs (Zappulli et al. 2005; Johri et al. 2006).
In Malaysia, outbreaks of Strep. agalactiae were mostly reported in cultured red hybrid tilapia (Oreochromis sp.) during the hot and dry seasons, causing severe economic losses to the farm operators (Najiah et al. 2009; Abuseliana et al. 2010; Amal et al. 2010a,b; Zamri-Saad et al. 2010). Recent studies in this country showed that high prevalence of Strep. agalactiae infection in red hybrid tilapia was observed in floating net cage cultures located in lakes and rivers, compared to earthern ponds, ex-mining pools and irrigation canals. Mortality rates were significantly higher during April to September, which were the most critical months of the year, affecting fish weighing between 100 and 300 g (Amal 2011).
Molecular typing techniques are specifically useful for epidemiological studies, as they provide information on the genetic relatedness of strains, sources of infection, detection of particularly virulent strains and the geographical and host distribution of possible variants of a specific pathogen (Olive and Bean 1999). Several genotyping techniques such as multi-locus enzyme electrophoresis (MLEE) (Quentin et al. 1995), pulsed field gel electrophoresis (PFGE) (Pereira et al. 2010; Chen et al. 2012), amplified fragment length polymorphism (Olivares-Fuster et al. 2008), single-stranded conformation polymorphism analysis of the intergenic spacer region (SSCP-ISR) (Olivares-Fuster et al. 2008) and multi-locus sequence typing (Al Nakib et al. 2011; Ye et al. 2011) have been utilized to characterize Strep. agalactiae from different host sources.
These techniques included several PCR-based techniques such as random amplified polymorphic DNA PCR (RAPD) and repetitive extragenic palindromic PCR (REP-PCR). The RAPD technique involves the use of short random sequence primers, usually 9 to 10 nucleotides long, and low-stringency primer annealing conditions to amplify arbitrary fragments of template DNA. The single primer anneals anywhere on the genome where a near-complementary sequence exists, and amplification occurs if two priming sites are sufficiently close (Welsh and McClelland 1990). However, REP-PCR technique utilizes primers that are complementary to specific sequences in the bacterial genome. The REP-PCR sequences consist of a highly conserved 33-bp inverted repeat sequence (Versalovic et al. 1991).
Previous studies have utilized RAPD and REP-PCR techniques in characterizing Strep. agalactiae strains isolated from various hosts, such as humans, bovines and fishes (Chatellier et al. 1997; Dodson et al. 1999; Martinez et al. 2000; Jafar et al. 2009; Nur-Nazifah et al. 2011). Using the RAPD technique, Jafar et al. (2009) successfully identified the sewage as the source of Strep. agalactiae infection in various fish species in Kuwait. Moreover, a study conducted by Nur-Nazifah et al. (2011) concluded that RAPD technique was a powerful method in differentiating Strep. agalactiae strains collected from different geographical locations. The results also proved that the PCR-based technique was valuable in epidemiological study, especially in disease traceability, transmission and prevention.
In this study, we explored the RAPD and REP-PCR techniques for the analysis of genetic variability within the Strep. agalactiae strains. We also established the DNA fingerprints of Strep. agalactiae strains from Malaysia along with evaluating the applicability of these techniques in epidemiological studies. As far as we are aware, this is the first study of this kind carried out on a large collection of Strep. agalactiae strains isolated from fishes in Malaysia.
Briefly, swab samples were collected from the body ulcer, brain, eye or kidney of diseased fish and streaked directly onto tryptic soy agar (TSA; Merck, Darmstadt, Germany) supplemented with 5% human blood and incubated for 24 h at 37°C. The dominant cultured bacteria were further subcultured to obtain pure colonies. All pure isolates were subjected to Gram stain, catalase and oxidase tests. Then, the Gram-positive, oxidase- and catalase-negative isolates were selected and subjected to API 20 STREP and Slidex Strepto-kits (bioMérieux, Marcy I'Etoile, France). Confirmation of Strep. agalactiae was made using PCR as previously described by Amal et al. (2012). All strains were maintained frozen at −80°C in brain–heart infusion (BHI; Merck) broth supplemented with 15% glycerol until used.
The code of the strains, geographical origin, number of sample, host species and year of isolation were described in Table 1. All strains were collected from diseased fish in floating net cage culture farms. The states of Kedah and Penang were located in the north, while Terengganu was in the east of Peninsular Malaysia (Fig. 1). For comparative purposes, Strep. agalactiae strain ATCC 27956 was used as a control.
|Code||Sampling site||State||Type of water body||No. of culture||Host species||Year of isolation|
|TPK||Pantai Kamlon||Penang||Ex-mining pool||1||Red hybrid tilapia (Oreochromis sp.)||2008|
|TJ||Jitra||Kedah||Earthen pond||5||Red hybrid tilapia (Oreochromis sp.)||2008|
|TK||Kodiang||Kedah||Irrigation canal||1||Red hybrid tilapia (Oreochromis sp.)||2007|
|TSB||Beladau Selat||Terengganu||River||22||Red hybrid tilapia (Oreochromis sp.)||2007–2008|
|TSK||Beladau Kepong||Terengganu||River||6||Red hybrid tilapia (Oreochromis sp.)||2007–2008|
|TSP||Pantai Ali||Terengganu||River||15||Red hybrid tilapia (Oreochromis sp.)||2007–2008|
|TKK||Kuala Kejir||Terengganu||River||2||Red hybrid tilapia (Oreochromis sp.)||2007|
|TP||Pedu Lake||Kedah||Reservoir||79||Red hybrid tilapia (Oreochromis sp.)||2007–2008|
|TCT||Kenyir Lake||Terengganu||Reservoir||47||Red hybrid tilapia (Oreochromis sp.)||2007–2008|
|BA||Bukit Tambun||Penang||Sea||3||Golden pompano (Trachinotus blochii)||2010|
All strains were grown on TSA (Merck) supplemented with 5% human blood and incubated for 24 h at 37°C. The DNA of Strep. agalactiae was extracted using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), in accordance with the manufacturer's protocol and maintained at −20°C until used. The extracted DNA was optimized for the concentration ranging between 3·5 and 4·0 μg μl−1 prior to PCR amplification.
Twenty 10-mer primers (OPA1 to OPA20) (Operon Technologies, Alameda, CA, USA) were tested for amplifications. The evaluation and selection of the primers was in accordance with the method described by Maluping et al. (2005). The amplifications were performed in a mixture of 25 μl, consisting of 1 μl genomic DNA of Strep. agalactiae, 1 μl primer (100 pmol μl−1) (NHK Bioscience, Kuala Lumpur, Malaysia), 2·5 μl 10× PCR buffer (Fermentas, Hanover, MD, USA), 2 μl MgCl2 (25 mmol l−1) (Fermentas), 0·5 μl dNTP (10 mmol l−1) (Fermentas), 0·5 μl Taq DNA Polymerase (5 μ μl−1) (Fermentas) and 17·5 μl of distilled water. A negative control of distilled water was included in each run to monitor for any contamination.
The amplifications were carried out in Pro S thermocycler machine (Eppendorf, Hamburg, Germany). The amplification started with 1 cycle of initial denaturation at 94°C for 2 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 36°C for 1 min and elongation at 72°C for 2 min. The amplification ended with a final elongation at 72°C for 5 min.
A published primer sequence of BOXA2R (Malathum et al. 1998) was used for amplification (Table 2). The amplification mixture used in this technique was similar to RAPD technique. A negative control of distilled water was included in each run to monitor for any contamination.
|Method||Primer||Sequence (5′–3′)||Size (bp)||% (G-C)||AT (°C)||Product size (bp)||No. of products|
|RAPD||OPA3||AGT CAG CCA C||10||60||36||200–3300||17|
|RAPD||OPA17||GAC CGC TTG T||10||60||36||250–2300||15|
|REP-PCR||BOXA2R||ACG TGG TTT GAA GAG ATT TTC G||22||41||40||210–2665||13|
The amplifications were carried out in Pro S thermocycler machine (Eppendorf). The amplification started with 1 cycle of initial denaturation at 95°C for 7 min, followed by 35 cycles of denaturation at 90°C for 30 s, annealing at 40°C for 1 min and elongation at 65°C for 8 min. The amplification ended with a final elongation at 65°C for 16 min.
One microlitre of the amplified products was mixed with 6× loading dye (Fermentas) and separated by agarose gel electrophoresis (1% agarose, in 1× TBE at 70 V, 400 mAmp for 1 h) in parallel with 100-bp and 1-kb DNA ladders (Fermentas) and stained with ethidium bromide. The DNA fragments were observed by UV transilluminator and photographed.
The banding pattern for each primer was scored using the Gene Tool software (Syngene, Cambridge, UK), and fragment sizes were estimated based on the DNA ladder mix. Meanwhile, alleles were designated based on fragment size; bands were scored as diallelic (1 = band present, 0 = band absent) and stored in Microsoft Office Excel spreadsheet (Microsoft, San Francisco, CA, USA) file as well as in NEXUS format.
The data analyses for both techniques were performed using NTSYS-pc (Exeter Software, Setauket, NY, USA) (Rohlf 2002), while the computed similarities among strains were estimated by means of the Dice coefficient (SD) (Dice 1945). For RAPD technique, data were analysed from the combination of both primer sets (OPA3 and OPA17). Dendrograms were constructed on the basis of the unweighted average pair group method. Due to very large number of samples in this study, the genetic groups were defined at a similarity level of 70% in order to allow for a better interpretation and understanding of the results.
The RAPD analysis of the Strep. agalactiae was performed on 30 randomly selected strains using 20 different primers (OPA1 to OPA20). Only two of them, OPA3 and OPA17 (Table 2), generated reproducible patterns with an appropriate number of amplified products (≥15) suitable for an accurate analysis. Analyses with the remaining primers did not result in a good amplification of the strains or provided only a small number of PCR products (≤15).
The selected primers, OPA3 and OPA17, were then used to analyse 181 strains of Strep. agalactiae. A set of reproducible bands produced for a particular primer was defined as a pattern or profile. The reproducibility of the RAPD technique was tested by repeating the RAPD assays twice for each primer tested. The results revealed that apart from some minor variations in the band intensity, no differences were detected between the obtained PCR products for both trials, which demonstrated the reproducibility of the method.
For RAPD technique, primer OPA3 produced a cumulative of 17 different bands from 182 strains tested (including the Strep. agalactiae strain ATCC 27956), with sizes ranging from 200 to 3300 bp, while each strain produced between two to nine bands. For OPA17, a cumulative of 15 different band sizes was produced with the range of 250 to 2300 bp, while each strain produced between one to nine bands. The example of banding patterns for OPA3 and OPA17 primers (showed only from one to eight bands), from 13 selected strains of different sampling sites, is presented in Fig. 2.
The constructed dendrogram from the RAPD technique divided all the strains into 13 different groups (Fig. 3). Groups 1, 2, 10, 11, 12 and 13 were recorded as the smallest group, composed of only one single strain (0·6%) per group, while the largest was group 4 with 70 strains (38·5%). Groups 3, 5, 6, 7, 8 and 9 were composed of 14 (7·7%), 55 (30·2%), 3 (1·6%), 7 (3·8%), 20 (11·0%) and 7 (3·8%) strains per group, respectively.
Groups 1 to 4 were only composed of strains from northern Peninsular Malaysia. The only strain isolated from red hybrid tilapia in Pantai Kamlon and Kodiang was composed of groups 1 and 2, respectively. Both the strains demonstrated a unique profile. Group 3 was composed of strains from Jitra and Pedu Lake, while all strains in group 4 were obtained from Pedu Lake.
Contrarily, the strains of groups 5 to 9 were dominated by strains from the east of Peninsular Malaysia. Group 5 was composed of strains from all the sites in Terengganu, while groups 6 and 8 were only represented by strains obtained from Beladau Kepong and Kenyir Lake, respectively. Similarly, groups 7 and 9 were only represented by strains obtained from Beladau Selat.
From this technique, the clustering analysis also revealed that three strains of Strep. agalactiae isolated from golden pompano, BA1, BA2 and BA5, showed a rather distinctive profile from each other and tend to cluster into groups 10, 11 and 12, respectively. The same result was also indentified for Strep. agalactiae strain ATCC 27956 in group 13.
The REP-PCR technique using the BOXA2R primer produced a cumulative of 13 different bands from 182 strains tested (including the Strep. agalactiae strain ATCC 27956), ranging from 210 to 2665 bp, while between two and nine bands were produced from each strain. The example of banding patterns for BOXA2R primer (showed only from three to nine bands), from 13 selected strains of different sampling sites, is presented in Fig. 2.
A total of 9 different genetic groups were produced using the REP-PCR technique (data not shown). A total of three groups (groups 1, 2 and 9) were recorded as the smallest group, composed of only one single strain (0·6%) per group. The largest group was group 5, with the total number of 72 strains (39·6%) per group. Groups 3, 4, 6, 7 and 8 were composed of 4 (2·2%), 8 (4·4%), 56 (30·8%), 36 (19·8%) and 3 (1·6%) strains per group, respectively.
Groups 1 to 5 were composed of only strains from northern Peninsular Malaysia. Similarly with RAPD technique, the only strain isolated from red hybrid tilapia in Pantai Kamlon and Kodiang displayed a unique profile and was composed of groups 1 and 2, respectively. Groups 3 and 4 were only composed of strains isolated from Jitra and Pedu Lake, respectively, while group 5 was composed of strains from Pedu Lake and a strain from Jitra.
All the strains from the east of Peninsular Malaysia were composed of either group 6 or 7. Group 6 was composed of all strains from the sampling sites in Terengganu, while for group 7, with the exception of Kuala Kejir, all strains were also composed from all the sampling sites in Terengganu too.
All strains of Strep. agalactiae recovered from golden pompano (BA1, BA2 and BA5) were located in group 8, while Strep. agalactiae strain ATCC 27956, which showed a unique profile, was the only strain in group 9.
Limited information was available on the epidemiology of Malaysian Strep. agalactiae strains recovered from fishes. To the best of our knowledge, no study using DNA-based techniques have been carried out with a large collection of field isolates of fishes origin in Malaysia.
In this study, both the REP-PCR and RAPD techniques were used as molecular tools to characterize Strep. agalactiae strains isolated from fishes in Malaysia. It was observed that these two fingerprinting techniques successfully generated reproducible band patterns, differing in number and molecular mass amplicons, which were suitable for an accurate analysis of Strep. agalactiae.
The RAPD and REP-PCR fingerprinting techniques have been widely used for molecular diagnosis for bacteria, due to its status as a powerful tool that enhances comparative analysis of genomes between different isolates of the same species or subspecies (O'hIci et al. 2000; Mukhopadhyay et al. 2001; Albufera et al. 2009; Labella et al. 2010; Nath et al. 2010). Previous studies successfully used both the RAPD and REP-PCR techniques for molecular characterization of Streptococcus sp. strains isolated from human, bovine, fish and sewage water samples (Chatellier et al. 1997; Dodson et al. 1999; Martinez et al. 2000; Jafar et al. 2009; Nur-Nazifah et al. 2011). Moreover, the PCR-based molecular typing methods, such as RAPD and REP-PCR, are fast, simple, sensitive, cost-efficient and practical for application in large number of samples and in the absent of high-technology equipments.
Based on the total number of strain groups produced in dendrogram analysis, this study clearly demonstrated that the combination of primers OPA3 and OPA17 for the RAPD technique produced higher discriminatory power compared to REP-PCR. This could be explained when the RAPD technique successfully divided 182 strains, including the Strep. agalactiae strain ATCC 27956, into 13 different groups, compared to 9 groups by REP-PCR. This finding was also supported by Chatellier et al. (1997), especially when they revealed that the combination of four primers (OPS16, AP42, A4 and OPS11) in a RAPD analysis produces higher discrimination index compared to when using a single primer in the characterization of Strep. agalactiae isolated from cerebrospinal fluid of humans.
It is interesting to note that the first of four and five groups from the dendrogram analyses of RAPD and REP-PCR, respectively, were dominated by strains of Strep. agalactiae isolated from red hybrid tilapia from the north (TPK, TK, TJ and TP), while the fifth to ninth and sixth to seventh of RAPD and REP-PCR analyses, respectively, were dominated by the strains from the east part of Peninsular Malaysia (TSB, TSK, TSP, TKK and TCT). Moreover, from both techniques, it was also discovered that several groups were only composed of strains from a specific sampling sites or geographical area, such as group 4 (Pedu Lake) for RAPD and group 3 (Jitra), group 4 (Pedu Lake) and group 5 (Pedu Lake, with the exception of strain TJ918) for REP-PCR. Interestingly, the availability of both techniques in differentiating the genetic profiles of the strains according to their geographical locations in this study is imperative, as it could be used in the future for disease traceability and determination of the source of Strep. agalactiae infections in Malaysia.
On the contrary, a study conducted by Nur-Nazifah et al. (2011) on 25 strains of Strep. agalactiae isolated from tilapia from five different locations in Malaysia using RAPD technique revealed that the genetic variation from different sampling locations were relatively high, even though the strains were collected from the same geographical region. Moreover, the authors also suggested that the main reasons for their observation might be due to the sources of the fish fry itself or the results of transposable elements, insertions or deletions in the DNA sequences of the obtained Strep. agalactiae strains.
Groups 5 and 6 for RAPD and REP-PCR analyses, respectively, were composed of mixed strains from all sampling sites in Terengganu. The high similarity of genetic profile (>70%) among the strains in this geographical location might suggest that all the strains may have come from the same source or hatchery and were recirculated in the fish culture system. Moreover, Amal (2011) observed that due to the active aquaculture activities and increasing demands for red tilapia fry, a single streptococcus-infected hatchery could possibly be responsible for transmitting Strep. agalactiae into several red hybrid tilapia farms from various geographical locations throughout the country. Recently, using RAPD and REP-PCR techniques, Amal et al. (2013) revealed that four strains of Strep. agalactiae collected from fish and water in a hatchery and a newly established red hybrid tilapia farm, respectively, showed identical profiles, indicating the isolated strains came from the same source. They believed that the strains were from the hatchery, and the pathogens were transferred together with the fish and water into the newly established farm.
Jafar et al. (2009) used RAPD technique in molecular investigation of Strep. agalactiae isolates from environmental samples and fish specimens during a massive fish kill in Kuwait Bay, Kuwait. Interestingly, they found that majority of the isolates recovered from mullet exhibited RAPD pattern that was highly similar to those obtained from strains recovered from sewage water, thereby implying their common origin.
Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains recovered from red hybrid tilapia. These results suggest that the genetics of Strep. agalactiae strains in this study was closely related to the host fish species, even though the results could also be influenced by the sample size, time of isolation and different type of environments where the strains were isolated. However, several previous studies revealed the relationship between the genetic specificity of bacterial fish pathogen and the host species. For example, Avendano-Herrera et al. (2004) found that the genetic variability among a species of fish pathogen, Tenacibaculum maritimum, which was isolated from several different countries, was strongly associated with the host fish species.
As expected, Strep. agalactiae strain ATCC 27956 recovered from bovine showed a unique profile for both methods. These results were not surprising, as previous study using PFGE method also revealed that strains from fish, bovine and human did not show genetic relatedness (Pereira et al. 2010). However, they proved that bovine and human strains were able to infect fish and caused meningoencephalitis, while a group of B Streptococcus serotype Ia isolated from a human neonatal meningitis patient recently caused disease and death of infected Nile tilapia (Evans et al. 2002; Pereira et al. 2010).
Dodson et al. (1999) used RAPD and REP-PCR techniques in the molecular typing of Streptococcus iniae isolated from cases of humans and fish. Even though the biochemical results from their study suggested that the fish and human Strep. iniae isolates were genetically different, the results of their RAPD and REP-PCR techniques showed lack of discriminatory abilities to differentiate between these Streptococcus isolates using four different primers of RAPD and BoxA primers for REP-PCR. Contradicting to their study, our results successfully revealed that both techniques possess excellent discriminative ability to differentiate the strains, even at the same host fish species and geographical location, indicating the reliability of primers and techniques used in this study.
In a study to compare and evaluate the effectiveness of RAPD and MLEE techniques in characterizing Strep. agalactiae isolated from cerebrospinal fluids samples from neonates, Chatellier et al. (1997) observed that RAPD analysis allowed further subdivision of strains that were not distinguishable by MLEE analysis. Using RAPD technique, the authors could identify the same virulent families in cluster analysis and also able to discriminate strains inside each cluster. Compared to MLEE technique, the produced virulent groups were apparently very homogeneous.
In summary, we have demonstrated the usefulness of RAPD and REP-PCR techniques for the analysis of genetic variability within the Strep. agalactiae strains isolated from fishes in Malaysia. The techniques tested were confirmed as excellent tools for molecular typing, along with possessing excellent discriminative ability and can be used as a rapid means of comparing Strep. agalactiae strains for epidemiological investigation. The results of this study were valuable as a guide for researchers studying disease control and prevention in the future. Moreover, it may help in the search for potential local vaccine candidates for streptococcosis, especially for this country. For future studies, it is recommended that the latest molecular technique be used when characterizing Strep. agalactiae strains.
The authors would like to thank Dr. Shabanimofrad M. and Mr. Mahdi E.M. for their technical assistance in preparing this manuscript, Bacteriology Laboratory from Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, UPM, for providing the Strep. agalactiae ATCC 27956 strain.